Peptides including amino acid sequences selected from lipoprotein (a) and apolipoprotein (a), antibodies recognizing these amino acid sequences, and methods of determination using these antibodies

ABSTRACT

Amino acid sequences having specificity as lipoprotein (a) and low homology with LDL and plasminogen are selected from the amino acid sequence of lipoprotein (a), and antibodies to lipoprotein (a) which recognize these amino acid sequences specifically are obtained. At least one of these antibodies is used in immunological techniques for the determination of lipoprotein (a). Moreover, an amino acid sequence having specificity as apolipoprotein (a) and not having antigenicity as lipoprotein (a) or plasminogen is selected from the amino acid sequence of apolipoprotein (a), and antibodies to apolipoprotein (a) which recognize this amino acid sequence specifically are obtained. At least one of these antibodies is used in immunological techniques for the determination of apolipoprotein (a). As compared with prior art methods, antibodies to lipoprotein (a) and apolipoprotein (a) can be obtained according to a simplified procedure, and their use makes it possible to determine lipoprotein (a) and apolipoprotein (a) accurately.

TECHNICAL FIELD

This invention relates to peptides selected from apolipoprotein (a) thatis a constituent of lipoprotein (a) being a risk factor involved inarteriosclerosis and having clinical significance, immunogens forproducing antibodies which recognize lipoprotein (a) or apolipoprotein(a) or peptides comprising constituent parts thereof, antibodiesrecognizing lipoprotein (a) or apolipoprotein (a) or peptides comprisingconstituent parts thereof, and methods for the determination oflipoprotein (a) or apolipoprotein (a) using these antibodies.

BACKGROUND ART

Lipoprotein (a) Lp(a)! was first reported in 1963 by Berg as a variantof β-lipoprotein K. Berg, Acta Pathol. Microbiol. Scand., 59,369-382(1963)!. As a result of subsequent various investigations, it hasbeen found that lipoprotein (a) is a substance consisting of low-densitylipoprotein (LDL) which usually plays a major role in transportingcholesterol esters within the living body, and of apolipoprotein (a) apo(a)! which is a protein peculiar to lipoprotein (a), the protein moietyof LDL (i.e., apolipoprotein B-100) and apolipoprotein (a) being linkedto each other by a disulfide bond (see FIG. 36).

Apolipoprotein (a) comprises at most 37 portions having high homologywith a structure, called Kringle 4, of plasminogen, a portion havinghigh homology with the Kringle 5 structure of plasminogen, and a serineprotease structure portion with a region having high homology withplasminogen. Of these 37 Kringle portions, 28 have a completelyrepeating structure.

Moreover, it has been reported that various isotypes (phenotypes) oflipoprotein (a) exist G. Utermann et al., J. Clin. Invest., 80,458-465(1987); H. G. Kraft et al., Arteriosclerosis, 8, 212-216(1988);G. Utermann et al., Hum. Genet., 78, 41-46(1988)!. This is considered tobe due to differences in the number of repetition of the Kringle4-corresponding portion of apolipoprotein (a) V. N. Trieu et al., J.Biol. Chem., 266, 5480-5485(1991)!. As a result of fractionation by acombination of SDS electrophoresis and an immunological blot technique,F, B, S1, S2, S3, S4 and O isotypes have been detected G. Utermann etal., J. Clin. Invest., 80, 458-465(1987)!.

Clinically, it has been found that the level of lipoprotein (a) is highin many patients with ischemic heart diseases such as angina pectorisand myocardial infarction G. Dahlen et al., Acta Med. Scand., (Suppl.)531, 1-29(1972); K. Berg et al., Clin. Genet., 16, 347-352(1979); G. M.Kostner et al., Atherosclerosis, 38, 51-61(1981)!. Moreover, it has beenreported that lipoprotein (a) shows no correlation with known riskfactors involved in ischemic heart diseases, such as cholesterol,LDL-cholesterol and HDL-cholesterol, and is a new independent riskfactor involved in arteriosclerosis and ischemic heart diseases C.Ehnholm et al., Biochim. Biophys. Acta, 236, 431-439(1971); H. Schrieweret al., J. Clin. Chem. Clin. Biochem., 22, 591-596(1984)!.

In 1987, Eaton et al. determined the amino acid sequence ofapolipoprotein (a) according to biochemical techniques D. L. Eaton etal., Proc. Natl. Acad. Sci. U.S.A., 84, 3224-3228(1987)!. Then, McLeanet al. determined it on the basis of the base sequence of cDNA J. W.McLean et al., Nature, 330, 132-137(1987)!.

Thus, it has been found that the greater part of the molecular structureof apolipoprotein (a) is composed of portions having high homology withthe plasminogen molecule acting on the fibrinolytic system.

This suggests that lipoprotein (a) and apolipoprotein (a) will providean important key when arteriosclerosis, lipoprotein and the bloodclotting and fibrinolytic system are considered from an identical pointof view.

Moreover, it has been reported that lipoprotein (a) is not only a riskfactor involved in arteriosclerosis, but also shows an increasingtendency in patients with diabetic nephropathy or reconstriction afterPTCA (percutaneous transluminal coronary angioplasty), and thatlipoprotein (a) behaves like acute phase reactive proteins such as CRP.Thus, the determination of lipoprotein (a) and apolipoprotein present inbiological samples such as blood has come to be of importantsignificance from a clinical point of view and, therefore, there is ademand for a method which enables accurate determination of lipoprotein(a) and apolipoprotein (a).

The determination of lipoprotein (a) is being made according toimmunological assay techniques such as single immunodiffusion, rocketimmunoelectrophoresis, turbidimetric immunoassay, latex turbidimetricimmunoassay, radioimmunoassay (RIA) J. J. Albers et al., J. Lipid Res.,18, 331-338(1977)! and enzyme immunoassay (EIA, ELISA) A. Abe et al.,Clin. Chim. Acta, 177, 31-40(1988)!. If an antibody prepared byadministering lipoprotein (a) as an immunogen to an animal is useddirectly in these immunological assay techniques, it is impossible toobtained accurate measured values for lipoprotein (a). The reason forthis is that, lipoprotein (a) contains LDL in its molecule and includesportions having high homology with plasminogen, the antibody reacts withany LDL and plasminogen present in biological samples and, therefore,LDL and plasminogen are measured together with lipoprotein (a).

Accordingly, there has been proposed a method which comprises preparingan antiserum (polyclonal antibody) to lipoprotein (a) by using, as animmunogen, apolipoprotein (a) obtained by removing the LDL moiety fromlipoprotein (a) G. M. Fless et al., J. Biol. Chem., 261,8712-8718(1986); G. Utermann et al., J. Biol. Chem., 265,981-986(1990)!. Although the antiserum (polyclonal antibody) obtained bythis method does not show a cross reaction with LDL, it has crossreactivity with plasminogen because apolipoprotein (a) contains aportion having high homology with plasminogen.

For that reason, this antiserum (polyclonal antibody) cannot be usedbefore it is subjected to a troublesome procedure for removingantibodies reacting with plasminogen by an absorption treatment withplasminogen.

Where apolipoprotein (a) is determined specifically (i.e., whereapolipoprotein (a) formed by breakage of the disulfide bond inlipoprotein (a) is determined), it is conceivable to prepare anantiserum (polyclonal antibody) by using apolipoprotein (a) as animmunogen and apply it to an immunological assay technique as describedabove.

However, since the antiserum (polyclonal antibody) prepared by usingapolipoprotein (a) as an immunogen also reacts with lipoprotein (a), itis necessary to remove therefrom an antibody reacting with lipoprotein(a) by an absorption treatment with lipoprotein (a), in addition to theabsorption treatment with human plasminogen. This is not onlytroublesome, but also disadvantageous in that it is difficult to obtainthe an antiserum (polyclonal antibody) in large amount because of itslow yield.

Moreover, the antisera and polyclonal antibodies to lipoprotein (a) orapolipoprotein (a) which are prepared in the above-described manner showvery great lot-to-lot variation. As a result, if determinations oflipoprotein (a) or apolipoprotein (a) are made under the sameconditions, the measured values may vary according to the lot of theantiserum or polyclonal antibody. Accordingly, it is necessary toreadjust the measuring conditions from lot to lot.

Also known is a method which comprises preparing a monoclonal antibodyto lipoprotein (a) or apolipoprotein (a) by using lipoprotein (a) orapolipoprotein (a) as an immunogen, and selecting a cell strainproductive of an antibody not reacting with LDL or plasminogen D. L.Eaton et al., Clin. Chem., 36, 192-197(1990); M. A. Lafferty et al., J.Lipid Res., 32, 277-292(1991)!. Among the antibody-producing cellstrains obtained in this method, however, there are only a few cellstrains productive of an antibody satisfying the requirements that theyshould be specific for lipoprotein (a) and should not show a crossreaction with LDL or plasminogen, or the requirements that they shouldbe specific for apolipoprotein (a) and should not show a cross reactionwith lipoprotein (a) or plasminogen. Thus, the selection of suchantibody-producing cell strains from a great number ofantibody-producing cell strains is an inefficient procedure requiringmuch labor and time.

Moreover, lipoprotein (a) and apolipoprotein (a), which are used asimmunogens for producing the above-described antiserum, polyclonalantibody and monoclonal antibody, must be obtained by purification frombiological samples. This purification procedure is delicate andtroublesome.

Furthermore, lipoprotein (a) and apolipoprotein (a) used as immunogenshave the disadvantage that they are not satisfactorily stable.Accordingly, even if they are purified through a troublesome procedure,it is difficult to store them for a long period of time.

In addition, when apolipoprotein (a) for use as an antibody-producingimmunogen is prepared by removing the LDL moiety from lipoprotein (a),apolipoprotein (a) may be denatured during the course of itspurification. Thus, it is very likely that the resulting product is notnative apolipoprotein (a).

Accordingly, the conventional antibodies to lipoprotein (a) orapolipoprotein (a) and the conventional immunogens for producing theseantibodies have the disadvantage that they require the above-describedcomplicated procedures for absorption treatment, for the correction oflot-to-lot variation, for the selection of an antibody-producing cellstrain, and for the purification of an immunogen, and involve muchlabor, time and cost.

DISCLOSURE OF THE INVENTION

In view of the above-described existing circumstances, the presentinventors made exhaustive investigations for the purpose of developingantibodies specifically recognizing lipoprotein (a) which do not show across reaction with LDL or plasminogen and can hence be obtained withoutrequiring troublesome procedures such as ones for absorption treatmentwith LDL or plasminogen, for the selection of a cell strain productiveof an antibody showing no cross reaction with LDL or plasminogen, forthe correction of lot-to-lot variation, and for the purification of animmunogen, and with less labor, time and cost as compared with prior artantibodies and methods, immunogens for producing these antibodies whichcan be obtained with less labor, time and cost as compared with priorart immunogens and methods, a method for the determination oflipoprotein (a) using these antibodies, and peptides selected from theamino acid sequence of lipoprotein (a) which peptides can be obtainedwith less labor, time and cost as compared with prior art peptides andmethods.

The present inventors also made exhaustive investigations for thepurpose of developing antibodies specifically recognizing apolipoprotein(a) which do not show a cross reaction with lipoprotein (a) orplasminogen and can hence be obtained without requiring troublesomeprocedures such as ones for absorption treatment with lipoprotein (a) orplasminogen, for the selection of a cell strain productive of anantibody showing no cross reaction with lipoprotein (a) or plasminogen,for the correction of lot-to-lot variation, and for the purification ofan immunogen, and with less labor, time and cost as compared with priorart antibodies and methods, immunogens for producing these antibodieswhich can be obtained with less labor, time and cost as compared withprior art immunogens and methods, a method for the determination ofapolipoprotein (a) using these antibodies, and peptides selected fromthe amino acid sequence of apolipoprotein (a) which peptides can beobtained with less labor, time and cost as compared with prior artpeptides and methods.

As a result, the present inventors have completed this invention.

1! SUMMARY OF THE INVENTION

The present invention includes the following inventions:

(1) A peptide composed of 50 or less amino acids and including a part orthe whole of the amino acid sequence which is selected from the aminoacid sequence of lipoprotein (a) and is represented by SEQ ID NO:1 orSEQ ID NO:2 in the List of Sequences.

(2) A peptide composed of 50 or less amino acids and including a part orthe whole of the amino acid sequence which is selected from the aminoacid sequence of apolipoprotein (a) and is represented by SEQ ID NO:3 inthe List of Sequences.

(3) An immunogen for producing an antibody to lipoprotein (a) whichcomprises a peptide composed of 50 or less amino acids, including a partor the whole of the amino acid sequence represented by SEQ ID NO:i inthe List of Sequences.

(4) An immunogen as described above in (3) wherein the peptide composedof 50 or less amino acids, including a part or the whole of the aminoacid sequence represented by SEQ ID NO:1 in the List of Sequences iscombined with a carrier.

(5) An immunogen for producing an antibody to lipoprotein (a) whichcomprises a peptide composed of 50 or less amino acids, including a partor the whole of the amino acid sequence represented by SEQ ID NO:2 inthe List of Sequences.

(6) An immunogen as described above in (5) wherein the peptide composedof 50 or less amino acids, including a part or the whole of the aminoacid sequence represented by SEQ ID NO:2 in the List of Sequences iscombined with a carrier.

(7) An immunogen for producing an antibody to apolipoprotein (a) whichcomprises a peptide composed of 50 or less amino acids, including a partor the whole of the amino acid sequence represented by SEQ ID NO:3 inthe List of Sequences.

(8) An immunogen as described above in (7) wherein the peptide composedof 50 or less amino acids, including a part or the whole of the aminoacid sequence represented by SEQ ID NO:3 in the List of Sequences iscombined with a carrier.

(9) A polyclonal antibody to lipoprotein (a) which specificallyrecognizes a part or the whole of the amino acid sequence represented bySEQ ID NO:1 in the List of Sequences.

(10) A polyclonal antibody as wherein the part (9) wherein the part ofthe amino acid sequence represented by SEQ ID NO:1 in the List ofSequences is the amino acid sequence represented by SEQ ID NO:4 in theList of Sequences.

(11) A polyclonal antibody as described above in (9) which is obtainedfrom an immunogen as described above in (3).

(12) An antibody to lipoprotein (a) which specifically recognizes a partor the whole of the amino acid sequence represented by SEQ ID NO:2 inthe List of Sequences.

(13) An antibody as described above in (12) wherein the part of theamino acid sequence represented by SEQ ID NO:2 in the List of Sequencesis the amino acid sequence represented by SEQ ID NO:5 in the List ofSequences.

(14) An antibody as described above in (12) which is obtained from animmunogen as described above in (5).

(15) An antibody as described above in (12), (13) or (14) which is amonoclonal antibody.

(16) An antibody to apolipoprotein (a) which specifically recognizes apart or the whole of the amino acid sequence represented by SEQ ID N0:3in the List of Sequences.

(17) An antibody as described above in (16) wherein the part of theamino acid sequence represented by SEQ ID NO:3 in the List of Sequencesis the amino acid sequence represented by SEQ ID NO:6 in the List ofSequences.

(18) An antibody as described above in (16) which is obtained from animmunogen as described above in (7).

(19) An antibody as described above in (16), (17) or (18) which is amonoclonal antibody.

(20) A method for the determination of lipoprotein (a) which comprisesusing at least one antibody as described above in any of (9) to (15).

(21) A method for the determination of apolipoprotein (a) whichcomprises using at least one antibody as described above in any of (16)to (19).

2! Selected amino acid sequences and peptides

In the present invention, the term "amino acid sequence selected fromthe amino acid sequence of lipoprotein (a)" refers to any amino acidsequence that is characterized by having specificity as lipoprotein (a)and low homology with LDL and plasminogen and that has been selectedfrom the amino acid sequence of lipoprotein (a).

Amino acid sequences having such characteristic features and peptidesincluding a part or the whole of such amino acid sequences exhibit thesame antigenicity as that possessed by lipoprotein (a) and do not havethe antigenicity of LDL or plasminogen. That is, they haveimmunogenicity capable of inducing the production of antibodiesrecognizing lipoprotein (a) specifically and can combine specificallywith antibodies to lipoprotein (a). Thus, they serve to determineantibodies recognizing lipoprotein (a) specifically and are henceuseful, for example, as immunogens for producing antibodies recognizinglipoprotein (a) specifically, as standard reference materials in thedetermination of lipoprotein (a) by an immunological assay technique, oras ligands in the purification of antibodies recognizing lipoprotein (a)specifically by affinity chromatography.

Also in the present invention, the term "amino acid sequence selectedfrom the amino acid sequence of apolipoprotein (a)" refers to any aminoacid sequence that is characterized by having specificity asapolipoprotein (a) and low homology with plasminogen and not havingantigenicity as lipoprotein (a) and that has been selected from theamino acid sequence of apolipoprotein (a).

Amino acid sequences having such characteristic features and peptidesincluding a part or the whole of such amino acid sequences exhibit thesame antigenicity as that possessed by apolipoprotein (a) and do nothave the antigenicity of lipoprotein (a) or plasminogen. That is, theyhave immunogenicity capable of inducing the production of antibodiesrecognizing apolipoprotein (a) specifically and can combine specificallywith antibodies to apolipoprotein (a). Thus, they serve to determineantibodies recognizing apolipoprotein (a) specifically and are henceuseful, for example, as immunogens for producing antibodies recognizingapolipoprotein (a) specifically, as standard reference materials in thedetermination of apolipoprotein (a) by an immunological assay technique,or as ligands in the purification of antibodies recognizingapolipoprotein (a) specifically by affinity chromatography.

When the amino acid sequences of the present invention satisfying thetwo requirements that (1) they should have specificity as lipoprotein(a) and (2) they should have low homology with LDL and plasminogen areselected from the amino acid sequence of lipoprotein (a), the amino acidsequence of apolipoprotein (a) must be used as a source in order toeliminate the participation of LDL.

Similarly, the amino acid sequence of the present invention satisfyingthe two requirements that (1) they should have specificity asapolipoprotein (a) and (2) they should not have antigenicity aslipoprotein (a) or plasminogen is also selected from the amino acidsequence of apolipoprotein (a).

It is desirable that such amino acid sequences are selected from anamino acid sequence repeated in apolipoprotein (a) as many times aspossible. The reason for this is that an antibody obtained by using apeptide including such an amino acid sequence as an antibody-producingimmunogen is expected to combine with lipoprotein (a) or apolipoprotein(a) in large numbers. This is advantageous in the determination oflipoprotein (a) or apolipoprotein (a) and, moreover, it is very likelythat such an antibody can provide accommodation for various isotypes oflipoprotein (a) or apolipoprotein (a).

Such an amino acid sequence is selected from the amino acid sequence ofapolipoprotein (a) that is composed of 4,529 amino acids.

The amino acid sequence so selected is divided into several segments andexamined.

When viewed from the standpoint of the stereostructure of protein, theamino acid sequence of a portion which has a high degree ofhydrophilicity, is very likely present on the surface of the proteinmolecule, is not contained in a special stereostructure and belongs to aflexible structure having a high degree of spatial wobble is consideredto be suitable for use as an amino acid sequence specificallyrepresenting the antigenicity of the substance used as anantibody-producing immunogen or the like.

Accordingly, the nature of each segment is estimated from thisviewpoint.

First of all, the degree of hydrophilicity of each amino acid residue isestimated according to the method of Hopp et al. T. P. Hopp et al.,Proc. Natl. Acad. Sci. U.S.A., 78, 3824-3828(1981)! and the method ofParker et al. Parker et al., Biochemistry, 25, 5425-5432(1986)!.

Moreover, according to the method of Garnier et al. Garnier et al., J.Mol. Biol., 120, 97-120(1987)!, an estimation is made to determinewhether each amino acid residue belongs to a special stereostructure.

Furthermore, according to the method of Karplus et al. Karplus et al.,Naturwissenschaften, 72, 212-213(1985)!, an estimation is made todetermine whether each amino acid residue belongs to a flexiblestructure having a high degree of spatial wobble.

In addition, reference may be made to the results of an investigation byScanu in which an estimation was made to determine the tendency for theamino acid residues of apolipoprotein (a) to assume an α-helix structureand a β-structure A. M. Scanu, "Lipoprotein (a)", Academic Press, SanDiego, 1990, p. 53-74!.

On the basis of the results thus obtained, segments having an amino acidsequence satisfying the above-described requirements are selected.

In order to obtain the present amino acid sequences selected from theamino acid sequence of lipoprotein (a), the amino acid sequences of thesegments selected as above are carefully compared with the amino acidsequence of plasminogen to find out amino acid sequences having lowhomology with the latter. Among these amino acid sequences, those havingsuitability for use as immunogens for producing an antibody tolipoprotein (a) are employed.

The amino acid sequences of the present invention which are representedby SEQ ID NO:1 and SEQ ID NO:2 in the List of Sequences have beenselected in the above-described manner.

In order to obtain the present amino acid sequence selected from theamino acid sequence of apolipoprotein (a), the amino acid sequences ofthe segments selected as above are carefully compared with the aminoacid sequence of plasminogen to find out amino acid sequences having lowhomology with the latter.

As the next step, in order to examine the antigenicity of these aminoacid sequences as lipoprotein (a), antibodies are prepared usingpeptides including these amino acid sequences as antibody-producingimmunogens, and the reactivity of the resulting antibodies withlipoprotein (a) is tested by the western blot technique or the like. Atthe same time, the reactivity of these antibodies with apolipoprotein(a) is tested by the western blot technique or the like.

On the basis of the finding thus obtained, an amino acid sequenceincluded in the immunogens of the antibodies not reacting withlipoprotein (a) is selected as an amino acid sequence having noantigenicity as lipoprotein (a).

The amino acid sequence of the present invention which is represented bySEQ ID NO:3 in the List of Sequences has been selected in theabove-described manner.

The present peptides composed of 50 or less amino acids and including apart or the whole of the amino acid sequence which is selected from theamino acid sequence of lipoprotein (a) and is represented by SEQ ID NO:1or SEQ ID NO:2 in the List of Sequences satisfy the two requirementsthat (1) they should have specificity as lipoprotein (a) and (2) theyshould have low homology with LDL and plasminogen, and exhibitantigenicity specific for lipoprotein (a) without exhibiting theantigenicity of LDL and plasminogen. Thus, they serve to determineantibodies recognizing lipoprotein (a) specifically and are henceuseful, for example, as immunogens for producing such antibodies, asstandard reference materials in the determination of lipoprotein (a),and as ligands in the purification of antibodies recognizing lipoprotein(a) specifically.

The present peptides composed of 50 or less amino acids and including apart or the whole of the amino acid sequence which is selected from theamino acid sequence of apolipoprotein (a) and is represented by SEQ IDNO:3 in the List of Sequences satisfy the two requirements that (1) theyshould have specificity as apolipoprotein (a) and (2) they should nothave antigenicity as lipoprotein (a) or plasminogen, and exhibitantigenicity specific for apolipoprotein (a) without exhibiting theantigenicity of lipoprotein (a) and plasminogen. Thus, they serve todetermine antibodies recognizing apolipoprotein (a) specifically and arehence useful, for example, as immunogens for producing such antibodies,as standard reference materials in the determination of apolipoprotein(a), and as ligands in the purification of antibodies recognizingapolipoprotein (a) specifically.

In the present peptides composed of 50 or less amino acids and includinga part or the whole of the amino acid sequence which is selected fromthe amino acid sequence of lipoprotein (a) and is represented by SEQ IDNO:1 or SEQ ID NO:2 in the List of Sequences, and the present peptidescomposed of 50 or less amino acids and including a part or the whole ofthe amino acid sequence which is selected from the amino acid sequenceof apolipoprotein (a) and is represented by SEQ ID NO:3 in the List ofSequences, the part of the amino acid sequence may be any sequence ofconsecutive amino acids included in the amino acid sequence representedby SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. Since there is a report thatan antibody can recognize an amino acid sequence composed of 3 aminoacids F. Hudecz et al., J. Immunol. Methods, 147, 201-210(1992)!, thepart of the amino acid sequence may preferably be any sequence of 3 ormore consecutive amino acids included in the amino acid sequencerepresented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3.

Moreover, the peptides composed of 50 or less amino acids and includinga part or the whole of the amino acid sequence represented by SEQ IDNO:1 or SEQ ID NO:2 in the List of Sequences are intended to cover, inaddition to peptides consisting of a part or the whole of the amino acidsequence represented by SEQ ID NO:1 or SEQ ID NO:2, such peptides havingone or more additional amino acid(s) or peptide(s) linked to theN-terminus or C-terminus, or both the N- and C-termini. No particularlimitation is placed on the amino acid(s) or peptide(s) so linked,provided that they do not include an amino acid sequence having highhomology with LDL or plasminogen. However, if the number of amino acidsis so increased as to form a large-sized peptide, homology with LDL orplasminogen may be produced and its stereostructure may be socomplicated as to cause the peptide to assume a special stereostructure.Accordingly, the peptides of the present invention should preferably becomposed of 50 or less amino acids, and more preferably 30 or less aminoacids.

Furthermore, the peptides composed of 50 or less amino acids andincluding a part or the whole of the amino acid sequence represented bySEQ ID NO:3 in the List of Sequences are intended to cover, in additionto peptides consisting of a part or the whole of the amino acid sequencerepresented by SEQ ID NO:3, such peptides having one or more additionalamino acid(s) or peptide(s) linked to the N-terminus or C-terminus, orboth the N- and C-termini. No particular limitation is placed on theamino acid(s) or peptide(s) so linked, provided that they do not includean amino acid sequence having high homology with plasminogen or aminoacid sequences having antigenicity as lipoprotein (a). However, if thenumber of amino acids is so increased as to form a large-sized peptide,homology with plasminogen or antigenicity as lipoprotein (a) may beproduced and its stereostructure may be so complicated as to cause thepeptide to assume a special stereostructure. Accordingly, the peptidesof the present invention should preferably be composed of 50 or lessamino acids, and more preferably 30 or less amino acids.

The present peptides composed of 50 or less amino acids and including apart or the whole of the amino acid sequence which is selected from theamino acid sequence of lipoprotein (a) and is represented by SEQ ID NO:1or SEQ ID NO:2 in the List of Sequences, and the present peptidescomposed of 50 or less amino acids and including a part or the whole ofthe amino acid sequence which is selected from the amino acid sequenceof apolipoprotein (a) and is represented by SEQ ID NO:3 in the List ofSequences, can be synthesized by any of various peptide synthesisprocesses including the liquid phase process and the solid phaseprocess, or by use of an automatic peptide synthesizer. For example,they can be synthesized according to any of the processes described inthe Japanese Biochemical Society (ed.) "Course of Lectures onBiochemical Experiments 1, Chemistry of Proteins IV", Tokyo KagakuDojin, 1975; Izumiya et al., "Foundation and Experimentation for PeptideSynthesis", Maruzen, 1985; and the Japanese Biochemical Society (ed.),"Course of Lectures on Biochemical ExpeChemiss (second series) 2,Chemistry of Proteins II", Tokyo Kagaku Dojin, 1987.

Alternatively, these peptides may be prepared from DNA having thecorresponding sequences according to the recombinant DNA technique. Forexample, such preparation can be carried out by reference to theJapanese Biochemical Society (ed.), "Course of Lectures on BiochemicalExperiments (second series) 1, Method for Gene Research I", Tokyo KagakuDojin, 1986; the Japanese Biochemical Society (ed.), "Course of Lectureson Biochemical Experiments (second series) 1, Method for Gene ResearchII", Tokyo Kagaku Dojin, 1986; and the Japanese Biochemical Society(ed.), "Course of Lectures on Biochemical Experiments (second series) 1,Method for Gene Research III", Tokyo Kagaku Dojin, 1987.

3! Antibody-producing immunogens

In the present immunogens for producing an antibody to lipoprotein (a)which comprise peptides composed of 50 or less amino acids, including apart or the whole of the amino acid sequence represented by SEQ ID NO:1or SEQ ID NO:2 in the List of Sequences, the peptides composed of 50 orless amino acids, including a part or the whole of the amino acidsequence represented by SEQ ID NO:1 or SEQ ID NO:2 in the List ofSequences comprise the above-described present peptides composed of 50or less amino acids and including a part or the whole of the amino acidsequence which is selected from the amino acid sequence of lipoprotein(a) and is represented by SEQ ID NO:1 or SEQ ID NO:2 in the List ofSequences.

These present immunogens for producing an antibody to lipoprotein (a)which comprise peptides composed of 50 or less amino acids, including apart or the whole of the amino acid sequence represented by SEQ ID NO:1or SEQ ID NO:2 in the List of Sequences make it possible to obtainantibodies satisfying the two requirements that (1) they shouldrecognize lipoprotein (a) specifically and (2) they should not show across reaction with LDL or plasminogen.

In the present immunogens for producing an antibody to apolipoprotein(a) which comprise peptides composed of 50 or less amino acids,including a part or the whole of the amino acid sequence represented bySEQ ID NO:3 in the List of Sequences, the peptides composed of 50 orless amino acids, including a part or the whole of the amino acidsequence represented by SEQ ID NO:3 in the List of Sequences comprisethe above-described present peptides composed of 50 or less amino acidsand including a part or the whole of the amino acid sequence which isselected from the amino acid sequence of apolipoprotein (a) and isrepresented by SEQ ID NO:3 in the List of Sequences.

These present immunogens for producing an antibody to apolipoprotein (a)which comprise peptides composed of 50 or less amino acids, including apart or the whole of the amino acid sequence represented by SEQ ID NO:3in the List of Sequences make it possible to obtain antibodiessatisfying the two requirements that (1) they should recognizeapolipoprotein (a) specifically and (2) they should not show a crossreaction with lipoprotein (a) or plasminogen.

In using the present immunogens for producing an antibody to lipoprotein(a) which comprise peptides composed of 50 or less amino acids,including a part or the whole of the amino acid sequence represented bySEQ ID NO:1 or SEQ ID NO:2 in the List of Sequences, and the presentimmunogens for producing an antibody to apolipoprotein (a) whichcomprise peptides composed of 50 or less amino acids, including a partor the whole of the amino acid sequence represented by SEQ ID NO:3 inthe List of Sequences, these peptides alone may be administered toanimals as antibody-producing immunogens, or these peptides may becombined with a carrier and then administered to animals asantibody-producing immunogens.

Where the immunogens are low-molecular-weight substances, it is commonto use them in a form combined with a carrier. However, there is areport that a peptide composed of 5 amino acids could be used as animmunogen to produce an antibody specific therefor (Kiyama et al.,Synopses of Lectures Given at the 112th Annual Meeting of thePharmaceutical Society of Japan, Vol. 3, 1992, P. 122). Accordingly, itis not essential to use a carrier.

Where it is desired to use a carrier, there may be used any ofwell-known carriers such as keyhole limpet hemocyanin (KLH), bovineserum albumin (BSA), human serum albumin, chicken serum albumin,poly-L-lysine, polyalanyllysine, dipalmityllysine, tetanus toxoid andpolysaccarides.

In order to combine the peptides of the present invention with carriers,there may be employed any of well-known methods such as theglutaraldehyde method, the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidemethod, the maleimidobenzoyl-N-hydroxysuccinimido ester method, theN-succinimidyl-3-(2-pyridyldithio)propionic acid method, thebisdiazotized benzidine method and the dipalmityllysine method.

Alternatively, the above-described peptides may be adsorbed onto acarrier such as nitrocellulose particles, polyvinyl pyrrolidone orliposomes and used as antibody-producing immunogens.

The present immunogens for producing an antibody to lipoprotein (a)which comprise peptides composed of 50 or less amino acids, including apart or the whole of the amino acid sequence represented by SEQ ID NO:1or SEQ ID NO:2 in the List of Sequences, and the present immunogens forproducing an antibody to apolipoprotein (a) which comprise peptidescomposed of 50 or less amino acids, including a part or the whole of theamino acid sequence represented by SEQ ID NO:3 in the List of Sequences,are stable because of their low molecular weights and can be stored fora long period of time.

4! Antibodies

The present polyclonal antibodies to lipoprotein (a) which specificallyrecognize a part or the whole of the amino acid sequence represented bySEQ ID NO:i in the List of Sequences, and the present antibodies tolipoprotein (a) which specifically recognize a part or the whole of theamino acid sequence represented by SEQ ID NO:2 in the List of Sequences,do not show a cross reaction with LDL or plasminogen, and can be used asantibodies recognizing lipoprotein (a) specifically.

The above-described polyclonal antibodies and antibodies to lipoprotein(a) have a specific affinity for a part or the whole of those amino acidsequences in lipoprotein (a). In other words, they have the property ofcombining specifically with a part or the whole of those amino acidsequences.

Thus, the above-described polyclonal antibodies and antibodies tolipoprotein (a) in accordance with the present invention can combinespecifically with not only lipoprotein (a), but also peptides orproteins including a part or the whole of the amino acid sequencerepresented by SEQ ID NO:1 or SEQ ID NO:2 in the List of Sequences,respectively,

In view of the fact that an antibody recognizes the antigenicdeterminants (or epitopes) of an antigen stereostructurally, it is to beunderstood that the term "a part of the amino acid sequence" as usedherein is not limited to a sequence of adjoining amino acids in theprimary structure of these amino acid sequences, but means anycombination of 3 or more amino acids in these amino acid sequences.

In connection with the above-described polyclonal antibodies andantibodies to lipoprotein (a) in accordance with the present invention,these amino acid sequences can further be reduced in size from theviewpoint of low homology with plasminogen. The amino acid sequencesthus obtained include the amino acid sequence represented by SEQ ID NO:4for the amino acid sequence represented by SEQ ID NO:2 in the List ofSequences, and the amino acid sequence represented by SEQ ID NO:5 forthe amino acid sequence represented by SEQ ID NO:2 in the List ofSequences.

Similarly, the present antibodies to apolipoprotein (a) whichspecifically recognize a part or the whole of the amino acid sequencerepresented by SEQ ID NO:3 in the List of Sequences do not show a crossreaction with lipoprotein (a) or plasminogen, and can be used asantibodies recognizing apolipoprotein (a) specifically.

The reason why the present antibodies to apolipoprotein (a) whichspecifically recognize a part or the whole of the amino acid sequencerepresented by SEQ ID NO:3 in the List of Sequences do not combine withlipoprotein (a) containing apolipoprotein (a) within its molecule, butspecifically recognize and combine with apolipoprotein (a) formed bybreakage of the disulfide bond in lipoprotein (a), is presumed to lie inthe differences in stereostructure, though it cannot be said definitelyat present.

The present antibodies to apolipoprotein (a) which specificallyrecognize a part or the whole of the amino acid sequence represented bySEQ ID NO:3 in the List of Sequences have a specific affinity for a partor the whole of this amino acid sequence in apolipoprotein (a). In otherwords, they have the property of combining specifically with a part orthe whole of this amino acid sequence.

Thus, the present antibodies to apolipoprotein (a) which specificallyrecognize a part or the whole of the amino acid sequence represented bySEQ ID NO:3 in the List of Sequences can combine specifically with notonly apolipoprotein (a), but also peptides or proteins including a partor the whole of the amino acid sequence represented by SEQ ID NO:3 inthe List of Sequences.

In view of the fact that an antibody recognizes the antigenicdeterminants (or epitopes) of an antigen stereostructurally, it is to beunderstood that the term "a part of the amino acid sequence" as usedherein is not limited to a sequence of adjoining amino acids in theprimary structure of this amino acid sequence, but means any combinationof 3 or more amino acids in this amino acid sequence.

In connection with the present antibodies to apolipoprotein (a) whichspecifically recognize a part or the whole of the amino acid sequencerepresented by SEQ ID NO:3 in the List of Sequences, this amino acidsequence can further be reduced in size from the viewpoint of lowhomology with plasminogen. The amino acid sequences thus obtainedinclude the amino acid sequence represented by SEQ ID NO:6.

The present polyclonal antibodies to lipoprotein (a) which specificallyrecognize a part or the whole of the amino acid sequence represented bySEQ ID NO:i in the List of Sequences, and the present antibodies tolipoprotein (a) which specifically recognize a part or the whole of theamino acid sequence represented by SEQ ID NO:2 in the List of Sequences,can be obtained by immunizing animals with an immunogen for producing anantibody to lipoprotein (a) which comprises a peptide composed of 50 orless amino acids, including a part or the whole of the amino acidsequence represented by SEQ ID NO:1 or SEQ ID NO:2 in the List ofSequences, respectively.

Similarly, the present antibodies to apolipoprotein (a) whichspecifically recognize a part or the whole of the amino acid sequencerepresented by SEQ ID NO:3 in the List of Sequences can be obtained byimmunizing animals with an immunogen for producing an antibody toapolipoprotein (a) which comprises a peptide composed of 50 or lessamino acids, including a part or the whole of the amino acid sequencerepresented by SEQ ID NO:3 in the List of Sequences.

The present polyclonal antibodies to lipoprotein (a) which specificallyrecognize a part or the whole of the amino acid sequence represented bySEQ ID NO:1 in the List of Sequences may be in the form of polyclonalantibodies themselves or antisera comprising polyclonal antibodies. Inaddition, fragments (such as Fab, F(ab')₂ and Fab') of these antibodiesare also fall within the scope of the present invention.

The present antibodies to lipoprotein (a) which specifically recognize apart or the whole of the amino acid sequence represented by SEQ ID NO:2in the List of Sequences, and the present antibodies to apolipoprotein(a) which specifically recognize a part or the whole of the amino acidsequence represented by SEQ ID NO:3 in the List of Sequences, may be inthe form of polyclonal antibodies, antisera comprising polyclonalantibodies, or monoclonal antibodies. In addition, fragments (such asFab, F(ab')₂ and Fab') of these antibodies are also fall within thescope of the present invention.

The polyclonal antibodies and antisera can be prepared according to thefollowing procedure.

First of all, a mammal (such as mouse, rabbit, rat, sheep, goat orhorse) or a bird (such as chicken) is immunized by administering theretothe antibody-producing immunogen which comprises a peptide composed of50 or less amino acids, including a part or the whole of the amino acidsequence represented by SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3 in theList of Sequences (either alone or in combination with a carrier).

The amount of antibody-producing immunogen administered may be suitablydetermined according to the type of the animal being immunized, the siteof injection, and the like. For example, in the case of mice, anantibody-producing immunogen containing the above-described peptide in asingle dose of 0.1 μg to 5 mg and preferably 50 μg to 1 mg per mouse isinjected into a mouse aged about 5 to 10 weeks. In the case of rabbits,it is preferable to inject an antibody-producing immunogen containingthe above-described peptide in a single dose of 10 μg to several tens ofmilligrams per rabbit.

Preferably, the antibody-producing immunogen is injected in admixturewith an adjuvant. As the adjuvant, there may be used any of well-knownadjuvants such as Freund's complete adjuvant, Freund's incompleteadjuvant, aluminum hydroxide adjuvant and pertussis adjuvant.

The immunizing injection may be given, for example, subcutaneously,intravenously, intraperitoneally or dorsally.

After the initial immunization, the antibody-producing immunogen isadditionally injected subcutaneously, intravenously, intraperitoneallyor dorsally as boosters at intervals of 2 to 3 weeks. Also in this case,the antibody-producing immunogen is preferably injected in admixturewith an adjuvant.

Following the initial immunization, the antibody titer in the bloodserum of the animal being immunized is repeatedly measured by ELISA orthe like. As soon as the antibody titer has reached a plateau,exsanguination is performed and blood serum is separated to obtain anantiserum.

This antiserum is purified by a purification technique such assalting-out with ammonium sulfate, sodium sulfate or the like, ionexchange chromatography, gel filtration or affinity chromatography, or acombination of such techniques. Thus, there can be obtained a polyclonalantibody in accordance with the present invention.

Where human serum albumin or BSA is used as a carrier for theantibody-producing immunogen, the resulting antibody or antiserum maycontain an antibody showing a cross reaction with human serum albumin.Accordingly, it is preferable to subject the resulting antibody orantiserum to a treatment for removing such an antibody. This removaltreatment may be accomplished, for example, by adding human serumalbumin or BSA used as a carrier to a solution of the resulting antibodyor antiserum and removing the aggregates so formed, or by subjecting theresulting antibody or antiserum to affinity chromatography in whichhuman serum albumin or BSA used as a carrier is immobilized on aninsoluble carrier.

Next, the method for the preparation of monoclonal antibodies isdescribed hereinbelow.

Monoclonal antibodies can be obtained by using antibody-producing cellssuch as hybridomas produced according to the cell fusion technique ofKoehler et al. G. Koehler et al., Nature, 256, 495-497(1975)!, or cellstumorigenically transformed by a virus such as Epstein-Barr virus.

The preparation of a monoclonal antibody according to the cell fusiontechnique can be carried out in the following manner.

First of all, a mammal such as mouse (e.g., of inbred strain BALB/c),nude mouse or rat! or a bird (such as chicken) is immunized byadministering thereto an antibody-producing immunogen which comprises apeptide composed of 50 or less amino acids, including a part or thewhole of the amino acid sequence represented by SEQ ID NO:2 or SEQ IDNO:3 in the List of Sequences. The amount of antibody-producingimmunogen administered may be suitably determined according to the typeof the animal being immunized, the site of injection, and the like. Forexample, in the case of mice, it is preferable to inject anantibody-producing immunogen containing the above-described peptide in asingle dose of 0.1 μg to 5 mg per mouse.

Preferably, the antibody-producing immunogen is injected in admixturewith an adjuvant. As the adjuvant, there may be used any of well-knownadjuvants such as Freund's complete adjuvant, Freund's incompleteadjuvant, aluminum hydroxide adjuvant and pertussis adjuvant.

The immunizing injection may be given, for example, subcutaneously,intravenously, intraperitoneally or dorsally.

After the initial immunization, the antibody-producing immunogen isadditionally injected subcutaneously, intravenously, intraperitoneallyor dorsally as boosters at intervals of 1 to 2 weeks. The number ofthese booster injections is commonly in the range of 2 to 6. Also inthis case, the antibody-producing immunogen is preferably injected inadmixture with an adjuvant.

Following the initial immunization, the antibody titer in the bloodserum of the animal being immunized is repeatedly measured by ELISA orthe like. As soon as the antibody titer has reached a plateau, a finalimmunization is performed by injecting a solution of theantibody-producing immunogen in physiological saline (a 0.9% aqueoussolution of sodium chloride) intravenously or intraperitoneally. Threeto five days after this final immunization, cells havingantibody-forming ability, such as spleen cells, lymph node cells orperipheral lymphocytes are collected from the immunized animal.

These antibody-forming cells obtained from the immunized animal andmyeloma cells of a mammal (such as mouse, nude mouse or rat) aresubjected to cell fusion. The myeloma cells are preferably of a cellstrain deficient in such an enzyme as hypoxanthine-guaninephosphoribosyltransferase (HGPRT) or thymidine kinase (TK). For example,there may be used the P3-X63-Ag8 (ATCC TIB9), P3-X63-Ag8-U1 JapaneseCancer Research Resources Bank (JCRB) 9085!, P3-NS1-1-Ag4-1 (JCRB 0009),P3-X63-Ag8. 653 (JCRB 0028) or SP2/0-Ag-14 (JCRB 0029) strains which areHGPRT-deficient cell strains derived from BALB/c mice.

The cell fusion can be effected with the aid of a fusion promoter suchas polyethylene glycols (PEG) having various molecular weights,liposomes or Sendai virus (HVJ), or by the electrical fusion method.

Where the myeloma cells are of a HGPRT- or TK-deficient strain, onlyfused cells (hybridomas) consisting of an antibody-forming cell and amyeloma cell can be selectively cultured and grown by using a screeningmedium containing hypoxanthine, aminopterin and thymidine (HAT medium).

By testing the supernatants obtained from the cultures of the resultinghydridomas according to an immunological assay technique such as ELISAor the western blot technique, a hybridoma productive of an antibody tolipoprotein (a) specifically recognizing a part or the whole of theamino acid sequence represented by SEQ ID NO:2 in the List of Sequencesor an antibody to apolipoprotein (a) specifically recognizing a part orthe whole of the amino acid sequence represented by SEQ ID NO:3 in theList of Sequences can be selected. Moreover, by using this procedure incombination with a well-known cloning technique such as limitingdilution, a cell strain productive of a monoclonal antibody inaccordance with the present invention can be isolated.

By culturing this monoclonal antibody-producing cell strain in asuitable medium, the monoclonal antibody of the present invention can beobtained from the supernatant of the culture thereof. A serum-freemedium or a low-serum medium may be used as the culture medium. Suchmedia are preferred because they facilitate purification of theantibody. For example, DMEM medium, RPMI1640 medium and ASF medium maybe used.

Alternatively, the monoclonal antibody-producing cell strain may beinjected into the abdominal cavity of a mammal which has compatibilitytherewith and has previously been stimulated by pristane or the like.After the laspe of a certain period of time, the monoclonal antibody ofthe present invention can be obtained from the ascites accumulated inthe abdominal cavity thereof.

The monoclonal antibody so produced can be purified by a purificationtechnique such as salting-out with ammonium sulfate, sodium sulfate orthe like, ion exchange chromatography, gel filtration or affinitychromatography, or a combination of such techniques. Thus, there can beobtained a purified monoclonal antibody in accordance with the presentinvention.

5! Methods of determination

The present method for the determination of lipoprotein (a) whichcomprises using at least one antibody selected from (1) a polyclonalantibody to lipoprotein (a) which specifically recognizes a part or thewhole of the amino acid sequence represented by SEQ ID NO:1 in the Listof Sequences, (2) a polyclonal antibody as described above in (1)wherein the part of the amino acid sequence represented by SEQ ID NO:1in the List of Sequences is the amino acid sequence represented by SEQID NO:4 in the List of Sequences, (3) a polyclonal antibody as describedabove in (1) that is obtained from an immunogen for producing antibodyto lipoprotein (a) which comprises a peptide composed of 50 or lessamino acids, including a part or the whole of the amino acid sequencerepresented by SEQ ID NO:1 in the List of Sequences, (4) an antibody tolipoprotein (a) which specifically recognizes a part or the whole of theamino acid sequence represented by SEQ ID NO:2 in the List of Sequences,(5) an antibody as described above in (4) wherein the part of the aminoacid sequence represented by SEQ ID NO:2 in the List of Sequences is theamino acid sequence represented by SEQ ID NO:5 in the List of Sequences,(6) an antibody as described above in (4) that is obtained from animmunogen for producing antibody to lipoprotein (a) which comprises apeptide composed of 50 or less amino acids, including a part or thewhole of the amino acid sequence represented by SEQ ID NO:2 in the Listof Sequences, and (7) an antibody as described above in (4), (5) or (6)which is a monoclonal antibody, can determine the concentration oflipoprotein (a) accurately without measuring together any LDL orplasminogen present in the samples.

Similarly, the present method for the determination of apolipoprotein(a) which comprises using at least one antibody selected from (1) anantibody to apolipoprotein (a) which specifically recognizes a part orthe whole of the amino acid sequence represented by SEQ ID NO:3 in theList of Sequences, (2) an antibody as described above in (1) wherein thepart of the amino acid sequence represented by SEQ ID NO:3 in the Listof Sequences is the amino acid sequence represented by SEQ ID NO:6 inthe List of Sequences, (3) an antibody as described above in (1) that isobtained from an immunogen for producing antibody to apolipoprotein (a)which comprises a peptide composed of 50 or less amino acids, includinga part or the whole of the amino acid sequence represented by SEQ IDNO:3 in the List of Sequences, and (4) an antibody as described above in(1), (2) or (3) which is a monoclonal antibody, can determine theconcentration of apolipoprotein (a) accurately without measuringtogether any lipoprotein (a) or plasminogen present in the samples.

In these methods of determination in accordance with the presentinvention, not only one of the above-described antibodies may be used,but also two or more of them may be used in combination.

The present methods of determination may be carried out according to anyassay technique using an antibody (i.e., any immunological assaytechnique), and can produce the desired effects by employing one of theabove-described antibodies as the antibody used in the technique. Forexample, the present methods of determination may be carried outaccording to any of various techniques such as enzyme immunoassay(ELISA, EIA), fluoroimmunoassay, radioimmunoassay (RIA),luminescentimmunoassay, enzyme-labeled antibody technique, fluorescent antibodytechnique, turbidimetric immunoassay, latex agglutination test, latexturbidimetric immunoassay, hemagglutination test, particle agglutinationand western blot technique.

The samples used in the present methods of determination can be anybiological samples that may contain lipoprotein (a) or apolipoprotein(a), or constituent parts thereof, such as blood, blood serum, bloodplasma, urine, cerebrospinal fluid, saliva, sweat, ascites, amnioticfluid, and cell or organ extracts.

Where the present methods of determination are carried out according toimmunoassays using a labeled antibody, such as enzyme immunoassay,fluoroimmunoassay, radioimmunoassay and luminescent immunoassay, eitherof the sandwich technique and the competitive assay technique may beemployed. In the sandwich technique, an antibody as described above maybe used as at least one of the antibodies combining directly withlipoprotein (a) or apolipoprotein (a), such as the capture antibody andthe labeled antibody.

As the solid-phase carrier, there may be used any of various well-knownsolid-phase carriers, for example, in the form of beads, microplates,test tubes, sticks and test strips made of such materials aspolystyrene, polycarbonate, polyvinyltoluene, polypropylene,polyethylene, polyvinyl chloride, nylon, polymethacrylate, latex,gelatin, agarose, cellulose, Sepharose, glass, metals, ceramics andmagnetic materials.

The capture antibody may be prepared from a solid-phase carrier and anantibody according to a well-known technique such as physicaladsorption, chemical bonding or a combination thereof.

The labeling material can be peroxidase (POD), alkaline phosphatase(ALP), β-galactosidase, urease, catalase, glucose oxidase, lactatedehydrogenase or amylase in the case of enzyme immunoassay; fluoresceinisothiocyanate, tetramethylrhodamine isothiocyanate, substitutedrhodamine isothiocyanate or dichlorotriazine isothiocyanate in the caseof fluoroimmunoassay; and tritium, iodine-125 or iodine-131 in the caseof radioimmunoassay. In luminescent immunoassay, there may be used anyof various systems such as NADH-FMNH₂ -luciferase, luminol-hydrogenperoxide-POD, acridinium ester and dioxetane compound systems.

In order to combine a labeling material with an antibody, there may beemployed any of well-known methods such as the glutaraldehyde method,the maleimide method, the pyridyl disulfide method and the periodic acidmethod.

Measurements can be made according to any of various well-knownprocesses The Japanese Society of Clinical Pathology (ed.), "ClinicalPathology (extra edition), No. 53, immunoassay for ClinicalExamination--Techniques and Applications--", Rinsho Byori Kankokai,1983; Eiji Ishikawa et al. (ed.), "Enzyme Immunoassay", Third Edition,Igaku Shoin, 1987; Tsunehiro Kitagawa et al. (ed.), "Proteins, NucleicAcids and Enzymes (extra issue), No. 31, Enzyme Immunoassay", KyoritsuShuppan, 1987!.

For example, the capture antibody is reacted with a sample, and furtherreacted with the labeled antibody simultaneously or after washing. Thus,a capture antibody-lipoprotein (a)-labeled antibody or captureantibody-apolipoprotein (a)-labeled antibody complex is formed. Bywashing and separating the unbound labeled antibody, the amount oflipoprotein (a) or apolipoprotein (a) in the sample can be determinedfrom the amount of the bound or unbound labeled antibody.

More specifically, in the case of enzyme immunoassay, a labeled enzymeis reacted with a substrate under the optimum conditions therefor, andthe amount of the reaction product is measured by optical means or thelike. The intensity of fluorescence produced by a fluorescent labelingmaterial is measured in fluoroimmunoassay, and the amount of radiationproduced by a radioactive labeling material is measured inradioimmunoassay. In the case of luminescent immunoassay, the amount oflight emitted from a luminescent reaction system is measured.

Where the present methods of determination are carried out by formingaggregates of an immune complex in turbidimetric immunoassay, latexagglutination test, latex turbidimetric immunoassay, hemagglutinationtest or particle agglutination or the like, and measuring thetransmitted or scattered light by optical means or visually, a phosphatebuffer, a glycine buffer, a Tris buffer or Good's buffer can be used asthe solvent and, moreover, a reaction promoter (e.g., polyethyleneglycol) or a nonspecific reaction inhibitor may be contained therein.

Where a solid-phase carrier is sensitized with an antibody, thesolid-phase carrier can be any of various particulate materialscomprising polystyrene, styrene-butadiene copolymers, (meth)acrylatepolymers, latex, gelatin, liposomes, microcapsules, erythrocytes,silica, alumina, carbon black, metallic compounds, metals, ceramics andmagnetic materials.

Such sensitization can be carried out according to a well-knowntechnique such as physical adsorption, chemical bonding or a combinationthereof.

Measurements may be made according to any well-known method. Forexample, where measurements are made by optical means, a sample isreacted with an antibody or a solid-phase carrier sensitized with anantibody, and the light transmitted or scattered thereby is measuredaccording to the end point or rate method.

Where measurements are made visually, a sample is reacted in a vesselsuch as a plate and a microplate with a solid-phase carrier sensitizedwith an antibody, and the degree of aggregation is evaluated visually.

Instead of visual evaluation, measurements may be made with the aid ofan instrument such as microplate reader.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the results of estimation of the degree ofhydrophilicity of each amino acid residue according to the method ofHopp et al.

FIG. 2 is a graph showing the results of estimation of the degree ofhydrophilicity of each amino acid residue according to the method ofParker et al.

FIG. 3 is a graph showing the results of estimation as to whether or noteach amino acid residue belongs to a special stereostructure, accordingto the method of Garnier et al.

FIG. 4 is a graph showing the results of estimation as to whether or noteach amino acid residue belongs to a flexible structure having a highdegree of spatial wobble, according to the method of Karplus et al.

FIG. 5 shows the results of analysis of the synthetic peptide obtainedin Example 3 by high-performance liquid chromatography (HPLC).

FIG. 6 shows the mass spectrum of the synthetic peptide obtained inExample 3.

FIG. 7 shows the results of analysis of the synthetic peptide obtainedin Example 4 by HPLC.

FIG. 8 shows the mass spectrum of the synthetic peptide obtained inExample 4.

FIG. 9 shows the results of analysis of the synthetic peptide obtainedin Example 5 by HPLC.

FIG. 10 shows the mass spectrum of the synthetic peptide obtained inExample 5.

FIG. 11 shows the results of ELISA carried out to examine the reactivityof the polyclonal antibody to lipoprotein (a) obtained in Example 9 withthe peptide represented by SEQ ID NO:8 in the List of Sequences.

FIG. 12 shows the results of ELISA carried out to examine the reactivityof the polyclonal antibody to lipoprotein (a) obtained in Example 10with the peptide represented by SEQ ID NO:9 in the List of Sequences.

FIG. 13 is a photograph showing electrophoretic patterns observed in thewestern blot technique carried out to examine the reactivity of thepolyclonal antibodies to lipoprotein (a) obtained in Examples 9 and 10with lipoprotein (a) and LDL.

FIG. 14 is a photograph showing electrophoretic patterns observed in thewestern blot technique carried out to examine the reactivity of thepolyclonal antibodies to lipoprotein (a) obtained in Examples 9 and 10with plasminogen.

FIG. 15 shows the results of ELISA carried out to examine the reactivityof the polyclonal antibody to apolipoprotein (a) obtained in Example 11with the peptide represented by SEQ ID NO:10 in the List of Sequences.

FIG. 16 is a photograph showing electrophoretic patterns observed in thewestern blot technique carried out to examine the reactivity of thepolyclonal antibody to apolipoprotein (a) obtained in Example 11 withapolipoprotein (a).

FIG. 17 is a photograph showing the electrophoretic patterns of anegative control observed in the western blot technique carried out toexamine the reactivity of the polyclonal antibody to apolipoprotein (a)obtained in Example 11 with apolipoprotein (a).

FIG. 18 is a photograph showing the electrophoretic pattern of a controlobserved in the western blot technique carried out to examine thereactivity of the polyclonal antibody to apolipoprotein (a) obtained inExample 11 with apolipoprotein (a).

FIG. 19 is a photograph showing electrophoretic patterns observed in thewestern blot technique carried out to examine the reactivity of thepolyclonal antibody to apolipoprotein (a) obtained in Example 11 withlipoprotein (a).

FIG. 20 is a graph showing a calibration curve for the determination oflipoprotein (a) by turbidimetric immunoassay using the polyclonalantibody to lipoprotein (a) obtained in Example 9.

FIG. 21 is a graph showing a calibration curve for the determination oflipoprotein (a) by turbidimetric immunoassay using the polyclonalantibody to lipoprotein (a) obtained in Example 10.

FIG. 22 is a graph showing a calibration curve for the determination oflipoprotein (a) by ELISA using the polyclonal antibody to lipoprotein(a) obtained in Example 9.

FIG. 23 is a graph showing a calibration curve for the determination oflipoprotein (a) by ELISA using the polyclonal antibody to lipoprotein(a) obtained in Example 10.

FIG. 24 is a graph showing the influence of plasminogen on the methodfor the determination of lipoprotein (a) in accordance with the presentinvention.

FIG. 25 is a graph showing a calibration curve for the determination ofapolipoprotein (a) by ELISA using the polyclonal antibody toapolipoprotein (a) obtained in Example 11.

FIG. 26 shows the results of ELISA carried out to examine the reactivityof the monoclonal antibody to lipoprotein (a) obtained in Example 22with the peptides represented by SEQ ID NO:8, SEQ ID NO:9 and SEQ IDNO:10 in the List of Sequences.

FIG. 27 shows the results of ELISA carried out to examine the reactivityof the monoclonal antibody to apolipoprotein (a) obtained in Example 23with the peptides represented by SEQ ID NO:8, SEQ ID NO:9 and SEQ IDNO:10 in the List of Sequences.

FIG. 28 is a photograph showing electrophoretic patterns observed in thewestern blot technique carried out to examine the reactivity of themonoclonal antibody to apolipoprotein (a) obtained in Example 23 withapolipoprotein (a).

FIG. 29 is a photograph showing the electrophoretic patterns of negativecontrols observed in the western blot technique carried out to examinethe reactivity of the monoclonal antibody to apolipoprotein (a) obtainedin Example 23 with apolipoprotein (a).

FIG. 30 is a photograph showing the electrophoretic pattern of a controlobserved in the western blot technique carried out to examine thereactivity of the monoclonal antibody to apolipoprotein (a) obtained inExample 23 with apolipoprotein (a).

FIG. 31 is a photograph showing electrophoretic patterns observed in thewestern blot technique carried out to examine the reactivity withlipoprotein (a) of the monoclonal antibody to lipoprotein (a) obtainedin Example 22 and the monoclonal antibody to apolipoprotein (a) obtainedin Example 23.

FIG. 32 is a photograph showing electrophoretic patterns observed in thewestern blot technique carried out to examine the reactivity with LDL ofthe monoclonal antibody to lipoprotein (a) obtained in Example 22 andthe monoclonal antibody to apolipoprotein (a) obtained in Example 23.

FIG. 33 is a photograph showing electrophoretic patterns observed in thewestern blot technique carried out to examine the reactivity withplasminogen of the monoclonal antibody to lipoprotein (a) obtained inExample 22 and the monoclonal antibody to apolipoprotein (a) obtained inExample 23.

FIG. 34 is a graph showing a calibration curve for the determination oflipoprotein (a) by ELISA using the monoclonal antibody to lipoprotein(a) obtained in Example 22.

FIG. 35 is a graph showing a calibration curve for the determination ofapolipoprotein (a) by ELISA using the monoclonal antibody toapolipoprotein (a) obtained in Example 23.

FIG. 36 is a schematic illustration of the structure of lipoprotein (a).

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention is more specifically described hereinbelow withreference to the following examples.

However, the present invention is in no way to be limited by theseexamples.

EXAMPLE 1

Selection of amino acid sequences from lipoprotein (a)

Amino acid sequences specifically representing the antigenicity oflipoprotein (a), which are characterized by having specificity aslipoprotein (a) and low homology with LDL and plasminogen, were selectedfrom the amino acid sequence of lipoprotein (a).

(1) In selecting amino acid sequences satisfying the two requirements(i.e., specificity as lipoprotein (a) and low homology with LDL andplasminogen) from the amino acid sequence of lipoprotein (a), the aminoacid sequence of apolipoprotein (a) was used as a source in order toeliminate the participation of LDL.

Next, the 4,529-amino acid sequence of apolipoprotein (a) J. W. McLeanet al., Nature, 330, 132-137(1987)! was examined to find out a sequencerepeated as many times as possible. Thus, there was selected an aminoacid sequence composed of 120 amino acids and represented by SEQ ID NO:7in the List of Sequences, which is a Kringle structure portion repeated28 times in the sequence extending from the 110th serine to the 3,306ththreonine as counted from the N-terminus of apolipoprotein (a).

(2) Employing cysteine residues as main criteria, this amino acidsequence represented by SEQ ID NO:7 was divided into 7 segments: segment1 extending from the 3rd alanine to the 14th cysteine as counted fromthe N-terminus, segment 2 extending from the 15th tyrosine to the 35thcysteine, segment 3 extending from the 36th glutamine to the 63rdcysteine, segment 4 extending from the 64th arginine to the 74thcysteine, segment 5 extending from the 75th tyrosine to the 86thcysteine, segment 6 extending from the 87th asparagine to the 91stcysteine, and segment 7 extending from the 92nd serine to the 116thglutamine.

(3) In order to learn the nature of these seven segments, the 120-aminoacid sequence represented by SEQ ID NO:7 was investigated in variousways.

The degree of hydrophilicity of each amino acid residue was estimatedaccording to the method of Hoppet al. T. P. Hopp et al., Proc. Natl.Acad. Sci. U.S.A., 78, 3824-3828(1981)!. The results of this estimationare shown in FIG. 1.

In FIG. 1, the abscissa indicates the serial number of amino acidresidue as counted from the N-terminus, while the ordinate indicates thedegree of hydrophilicity in which greater values represent higherdegrees of hydrophilicity and smaller values represent lower degrees ofhydrophilicity.

(4) Next, the degree of hydrophilicity of each amino acid residue wasestimated again according to the method of Parker et al. Parker et al.,Biochemistry, 25, 5425-5432(1986)!. The results of this estimation areshown in FIG. 2.

In FIG. 2, the abscissa indicates the serial number of amino acidresidue as counted from the N-terminus, while the ordinate indicates thedegree of hydrophilicity in which greater values represent higherdegrees of hydrophilicity.

(5) According to the method of Garnier et al. Garnier et al., J. Mol.Biol., 120, 97-120(1987)!, an estimation was made to determine whethereach amino acid residue belongs to a special stereostructure. Theresults of this estimation are shown in FIG. 3.

In FIG. 3, the abscissa indicates the serial number of amino acidresidue as counted from the N-terminus, while the ordinate indicates thetendency to assume a special stereostructure in which greater valuesrepresent greater tendencies to belong to a special stereostructure.

(6) According to the method of Karplus et al. Karplus et al.,Naturwissenschaften, 72, 212-213(1985)!, an estimation was made todetermine whether each amino acid residue belongs to a flexiblestructure having a high degree of spatial wobble. The results of thisestimation are shown in FIG. 4.

In FIG. 4, the abscissa indicates the serial number of amino acidresidue as counted from the N-terminus, while the ordinate indicates thetendency to assume a flexible structure in which greater valuesrepresent greater tendencies to belong to a flexible structure having ahigh degree of spatial wobble.

(7) In addition, reference was made to the results of an investigationby Scanu in which an estimation was made to determine the tendency foreach amino acid residue in the amino acid sequence represented by SEQ IDNO:7 to assume an α-helix structure and a β-structure A. M. Scanu,"Lipoprotein (a)", Academic Press, San Diego, 1990, p. 53-74!.

(8) As a result of examination of the foregoing data, it was thoughtthat segment 2 is very likely to assume a β-structure which is a specialstereostructure, and segment 5 is very likely to be buried in theinterior of the protein molecule because of its high degree ofhydrophobicity. Accordingly, the amino acid sequences of these twosegments were considered to be unsuitable for use as amino acidsequences specifically representing the antigenicity as anantibody-producing immunogen or the like.

Moreover, segment 6 is not suitable for use as an amino acid sequencespecifically representing antigenicity because a sugar is linkedthereto, and segment 3 cannot be used because its amino acid sequencehas high homology with the amino acid sequence of plasminogen.

(9) Accordingly, it was concluded that the present amino acid sequencesspecifically representing the antigenicity of lipoprotein (a), which arecharacterized by having specificity as lipoprotein (a) and low homologywith LDL and plasminogen, should preferably selected from segment 1,segment 4 and segment 7. As a result of close investigation made so asto minimize homology with the amino acid sequence of plasminogen and soas to be suitable for use as immunogens for producing antibody tolipoprotein (a), the amino acid sequences represented by SEQ ID NO:1 andSEQ ID NO:2 in the List of Sequences were selected.

EXAMPLE 2

Selection of an amino acid sequence from apolipoprotein (a)

An amino acid sequence specifically representing the antigenicity ofapolipoprotein (a), which is characterized by having specificity asapolipoprotein (a) and no antigenicity as lipoprotein (a) orplasminogen, was selected from the amino acid sequence of apolipoprotein(a).

(1) The 4,529-amino acid sequence of apolipoprotein (a) J. W. McLean etal., Nature, 330, 132-137(1987)! was examined to find out a sequencerepeated as many times as possible. Thus, there was selected an aminoacid sequence composed of 120 amino acids and represented by SEQ ID NO:7in the List of Sequences, which is a Kringle structure portion repeated28 times in the sequence extending from the 110th serine to the 3,306ththreonine as counted from the N-terminus of apolipoprotein (a).

(2) Employing cysteine residues as main criteria, this amino acidsequence represented by SEQ ID NO:7 was divided into 7 segments: segment1 extending from the 3rd alanine to the 14th cysteine as counted fromthe N-terminus, segment 2 extending from the 15th tyrosine to the 35thcysteine, segment 3 extending from the 36th glutamine to the 63rdcysteine, segment 4 extending from the 64th arginine to the 74thcysteine, segment 5 5 extending from the 75th tyrosine to the 86thcysteine, segment 6 extending from the 87th asparagine to the 91stcysteine, and segment 7 extending from the 92nd serine to the 116thglutamine.

In order to learn the nature of these seven segments, the 120-amino acidsequence represented by SEQ ID NO:7 was investigated in various ways.This was accomplished in the same manner as described in steps (3) to(7) of Example 1.

(8) As a result of examination of the foregoing data, it was thoughtthat segment 2 is very likely to assume a b-structure which is a specialstereostructure, and segment 5 is very likely to be buried in theinterior of the protein molecule because of its high degree ofhydrophobicity. Accordingly, the amino acid sequences of these twosegments were considered to be unsuitable for use as amino acidsequences specifically representing the antigenicity as anantibody-producing immunogen or the like.

Moreover, segment 6 is not suitable for use as an amino acid sequencespecifically representing antigenicity because a sugar is linkedthereto, and segment 3 cannot be used because its amino acid sequencehas high homology with the amino acid sequence of plasminogen.

(9) Accordingly, it was concluded that the present amino acid sequencespecifically representing the antigenicity of apolipoprotein (a) shouldpreferably selected from segment 1, segment 4 and segment 7. As a resultof close investigation made so as to minimize homology with the aminoacid sequence of plasminogen, the amino acid sequences represented bySEQ ID NO:1, SEQ ID NO:2 and SEQ ID NO:3 in the List of Sequences wereselected as candidates.

(10) Using a Model 430A peptide synthesizer (manufactured by AppliedBiosystems) according to its instruction manual, peptides including eachof these amino acid sequences represented by SEQ ID NO:1, SEQ ID NO:2and SEQ ID NO:3 in the List of Sequences were synthesized by thet-butoxycarbonylamino acid solid-phase method.

(11) The peptides so synthesized were combined with a carrier comprisingkeyhole limpet hemocyanin (KLH) (manufactured by Calbiochem) to prepareantibody-producing immunogens. Then, female BALB/c mice (Charles RiverJapan Inc.) aged 8 weeks were immunized with these immunogens to preparepolyclonal antibodies.

(12) The reactivity of these polyclonal antibodies with lipoprotein (a)was tested by the western blot technique using a Titan Gel LipoproteinElectrophoresis Kit (manufactured by Helena Laboratory) and a Nova BlotElectrophoretic Transfer Kit (manufactured by Pharmacia-LKB), with theresult that the polyclonal antibody prepared from the peptide includingthe amino acid sequences represented by SEQ ID NO:3 in the List ofSequences did not react with lipoprotein (a).

(13) At the same time, the reactivity of these polyclonal antibodieswith apolipoprotein (a) was tested by the western blot technique using4% SDS-polyacrylamide gel (manufactured by Tefco) and a Nova BlotElectrophoretic Transfer Kit (manufactured by Pharmacia-LKB). Thus, itwas found that all of these antibodies reacted with apolipoprotein (a).

(14) Accordingly, the amino acid sequence represented by SEQ ID NO:3 inthe List of Sequences was selected as the resent amino acid sequencespecifically representing the antigenicity of apolipoprotein (a), whichis characterized by having specificity as apolipoprotein (a) and noantigenicity as lipoprotein (a) or plasminogen.

EXAMPLE 3

Synthesis of the peptide represented by SEQ ID NO:8 in the List ofSequences

The peptide represented by SEQ ID NO:8 in the List of Sequences, whichis a peptide including the amino acid sequence represented by SEQ IDNO:1 in the List of Sequences, was synthesized.

First of all, using a Model 430A peptide synthesizer (manufactured byApplied Biosystems) according to its instruction manual, the peptide wassynthesized by the t-butoxycarbonylamino acid solid-phase method. Thesynthesized peptide was desorbed from the resin by the hydrogen fluoridemethod in the presence of dimethyl sulfide, p-thiocresol, m-cresol andanisole used as scavengers for suppressing side reactions.

Thereafter, the scavengers were extracted with dimethyl ether, and thesynthesized peptide was extracted with 2N acetic acid.

The extract was purified by anion exchange column chromatography usingthe anion exchange resin DOWEX 1-X2, and the pattern of a main peak wasconfirmed by high-performance liquid chromatography (HPLC) using anoctadecyl (ODS) column.

After the resulting peptide solution was concentrated by freeze-dryingin an evaporator, the peptide was purified and separated by HPLC. Theapparatus and conditions employed for this purification by HPLC were asfollows: Using the reverse phase ODS column YMC-D-ODS-5 (20 mm×300 mm;manufactured by Yamamura Chemical Laboratory), the peptide was elutedwith a gradient of 0% to 70% acetonitrile in 0.1% trifluoroacetic acid(TFA) at a flow rate of 7.0 ml/min. by means of a TWINCLE pump(manufactured by Jasco Corp.) and a Model GP-A40 gradienter(manufactured by Jasco Corp.) and detected by means of a ModelUVIDEC-100V detector (210 nm, 1.28 AUFS; manufactured by Jasco Corp.).

The synthetic peptide purified and separated in this manner wasconcentrated by freeze-drying in an evaporator.

The purity of the resulting synthetic peptide was analyzed by HPLC. Theapparatus and conditions employed for this purpose were as follows:Using the reverse phase ODS column YMC-R-ODS-5 (4.9 mm×300 mm;manufactured by Yamamura Chemical Laboratory), the peptide was elutedwith a gradient of 0% to 70% acetonitrile in 0.1% trifluoroacetic acid(TFA) at a flow rate of 1.0 ml/min. for 25 minutes by means of a58-59TWINCLE pump (manufactured by Jasco Corp.) and a Model GP-A40gradienter (manufactured by Jasco Corp.) and detected by means of aModel UVIDEC-100V detector (210 nm, 1.28 AUFS; manufactured by JascoCorp.).

The results of this analysis are shown in FIG. 5. In FIG. 5, PKNOdesignates the number of each peak in the chart, TIME designates theelution time, AREA designates the peak area, and CONC designates theproportion of that peak area to the total peak area (i.e., the percentconcentration).

It can be seen from these results that the purity of the resultingsynthetic peptide is 100%.

Moreover, the amino acid composition analysis of the resulting syntheticpeptide was made by using a Waters Pico-Tag amino acid analyzer(manufactured by Millipore) according to its instruction manual. Thepeptide sample was hydrolyzed by boiling in 6N hydrochloric acidcontaining 1% phenol at 150° C. for an hour.

The results of this amino acid composition analysis are shown inTable 1. (Since cysteine cannot be determined by hydrolysis withhydrochloric acid, its analytical value is omitted.)

                  TABLE 1                                                         ______________________________________                                                      Number of amino acid residues                                   Amino acid    in synthesized peptide                                          residue       Theoretical value                                                                         Found value                                         ______________________________________                                        Asx           1           1.0                                                 Glx           1           1.0                                                 Ser           1           0.9                                                 Gly           1           1.0                                                 Thr           1           1.0                                                 Ala           2           2.0                                                 Val           1           1.1                                                 ______________________________________                                    

In Table 1, Asx designates asparagine or aspartic acid, and Glxdesignates glutamine or glutamic acid.

From these results, it has been confirmed that the resulting syntheticpeptide has the same composition as the amino acid sequence representedby SEQ ID NO:8 in the List of Sequences and, therefore, is the peptiderepresented by SEQ ID NO:8 in the List of Sequences. The resultingsynthetic peptide had an isoelectric point of 2.9. Its mass spectrum isshown in FIG. 6.

EXAMPLE 4

Synthesis of the peptide represented by SEQ ID NO:9 in the List ofSequences

The peptide represented by SEQ ID NO:9 in the List of Sequences, whichis a peptide including the amino acid sequence represented by SEQ IDNO:2 in the List of Sequences, was synthesized.

Synthesis, purification and analysis were carried out in the same manneras in Example 3.

The purity of the resulting synthetic peptide was analyzed by HPLC andthe results thus obtained are shown in FIG. 7.

In FIG. 7, PKNO designates the number of each peak in the chart, TIMEdesignates the elution time, AREA designates the peak area, and CONCdesignates the proportion of that peak area to the total peak area(i.e., the percent concentration).

It can be seen from these results that the purity of the resultingsynthetic peptide is approximately 100%.

Moreover, the results of amino acid composition analysis of theresulting synthetic peptide are shown in Table 2.

                  TABLE 2                                                         ______________________________________                                                      Number of amino acid residues                                   Amino acid    in synthesized peptide                                          residue       Theoretical value                                                                         Found value                                         ______________________________________                                        Glx           5           4.8                                                 Ser           1           1.0                                                 Arg           1           1.1                                                 Thr           1           1.0                                                 Ala           2           2.0                                                 Pro           2           2.1                                                 ______________________________________                                    

In Table 2, Glx designates glutamine or glutamic acid.

From these results, it has been confirmed that the resulting syntheticpeptide has the same composition as the amino acid sequence representedby SEQ ID NO:9 in the List of Sequences and, therefore, is the peptiderepresented by SEQ ID NO:9 in the List of Sequences. The resultingsynthetic peptide had an isoelectric point of 4. its mass spectrum isshown in FIG. 8.

EXAMPLE 5

Synthesis of the peptide represented by SEQ ID NO:10 in the List ofSequences

The peptide represented by SEQ ID NO:10 in the List of Sequences, whichis a peptide including the amino acid sequence represented by SEQ IDNO:3 in the List of Sequences, was synthesized.

Synthesis, purification and analysis were carried out in the same manneras in Example 3.

The purity of the resulting synthetic peptide was analyzed by HPLC andthe results thus obtained are shown in FIG. 9.

In FIG. 9, PKNO designates the number of each peak in the chart, TIMEdesignates the elution time, AREA designates the peak area, and CONCdesignates the proportion of that peak area to the total peak area(i.e., the percent concentration).

It can be seen from these results that the purity of the resultingsynthetic peptide is approximately 100%.

Moreover, the results of amino acid composition analysis of theresulting synthetic peptide are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                                      Number of amino acid residues                                   Amino acid    in synthesized peptide                                          residue       Theoretical value                                                                         Found value                                         ______________________________________                                        Asx           2           2.0                                                 Arg           1           1.0                                                 Ala           3           2.7                                                 Pro           2           2.0                                                 Val           1           1.0                                                 ______________________________________                                    

In Table 3, Asx designates asparagine or aspartic acid.

From these results, it has been confirmed that the resulting syntheticpeptide has the same composition as the amino acid sequence representedby SEQ ID NO:10 in the List of Sequences and, therefore, is the peptiderepresented by SEQ ID NO:10 in the List of Sequences. The resultingsynthetic peptide had an isoelectric point of 5. Its mass spectrum isshown in FIG. 10.

EXAMPLE 6

Preparation of an antibody-producing immunogen comprising a carrierhaving combined therewith the peptide represented by SEQ ID NO:8 in theList of Sequences

10 mg of a carrier comprising keyhole limpet hemocyanin (KLH)(manufactured by Calbiochem) or bovine serum albumin (BSA) (manufacturedby Seikagaku Corp.) was dissolved in 10 mM potassium dihydrogenphosphate-dipotassium hydrogen phosphate buffer (pH 7.0), to which wasadded 150 μl of a 2.5% solution ofmaleimidobenzoyl-N-hydroxysuccinimideester (MBS) (manufactured byPierce) in N,N-dimethylformamide. This mixture was reacted at roomtemperature for 30 minutes with stirring.

This mixture was applied to a gel filtration column of Sephadex G-25(manufactured by Pharmacia-LKB) which was placed at 4° C. and had beenequilibrated with 10 mM potassium dihydrogen phosphate-dipotassiumhydrogen phosphate buffer (pH 7.0), and a combined MBS-carrier componentwas collected by monitoring the absorbance at 280 nm.

This combined MBS-carrier component was adjusted to pH 7.0 withtrisodium phosphate, and the peptide synthesized in Example 3 andrepresented by SEQ ID NO:8 in the List of Sequences was added theretoand mixed therewith. This mixture was reacted for 150 minutes.

After completion of the reaction, the mixture was dialyzed three timesagainst water and then freeze-dried to obtain an antibody-producingimmunogen comprising a carrier having combined therewith the peptiderepresented by SEQ ID NO:8 in the List of Sequences.

The yield was 89% when the carrier was KLH, and 67% when the carrier wasBSA.

The proportion (weight ratio) of the peptide represented by SEQ ID NO:8in the List of Sequences to the antibody-producing immunogen was 33%when the carrier was KLH, and 27% when the carrier was BSA.

EXAMPLE 7

Preparation of an antibody-producing immunogen comprising a carrierhaving combined therewith the peptide represented by SEQ ID NO:9 in theList of Sequences

Using the peptide synthesized in Example 4, an antibody-producingimmunogen comprising a carrier having combined therewith the peptiderepresented by SEQ ID NO:9 in the List of Sequences was prepared in thesame manner as in Example 6.

The yield was 73% when the carrier was KLH, and 64% when the carrier wasBSA.

The proportion (weight ratio) of the peptide represented by SEQ ID NO:9in the List of Sequences to the antibody-producing immunogen was 23%when the carrier was KLH, and 25% when the carrier was BSA.

EXAMPLE 8

Preparation of an antibody-producing immunogen comprising a carrierhaving combined therewith the peptide represented by SEQ ID NO:10 in theList of Sequences

Using the peptide synthesized in Example 5, an antibody-producingimmunogen comprising a carrier having combined therewith the peptiderepresented by SEQ ID NO:10 in the List of Sequences was prepared in thesame manner as in Example 6.

The yield was 75% when the carrier was KLH, and 52% when the carrier wasBSA.

The proportion (weight ratio) of the peptide represented by SEQ ID NO:10in the List of Sequences to the antibody-producing immunogen was 30%when the carrier was KLH, and 21% when the carrier was BSA.

EXAMPLE 9

Preparation of a mouse polyclonal antibody to lipoprotein (a)specifically recognizing the amino acid sequence represented by SEQ IDNO:i in the List of Sequences

The antibody-producing immunogen (having KLH as the carrier) obtained inExample 6 was dissolved in physiological saline (a 0.9% aqueous solutionof sodium chloride) so as to give a concentration of 360 μg/ml. Thissolution was mixed with an equal amount of Freund's complete adjuvant toform an emulsion, and 0.5 ml of this emulsion was subcutaneouslyinjected into the abdomen of a female BALB/c mouse (Charles River JapanInc.) aged 8 weeks for purposes of immunization.

Two weeks after the initial immunization, the above-describedantibody-producing immunogen was dissolved in physiological saline so asto give a concentration of 180 μg/ml. This solution was mixed with anequal amount of Freund's incomplete adjuvant to form an emulsion, and0.5 ml of this emulsion was injected as a booster. This boosterinjection was repeated at intervals of 2 weeks.

The antibody titer in the blood serum of this mouse, which was animmunized animal, was measured by an enzyme immunoassay (ELISA, EIA) atintervals of one week.

This ELISA was carried out as follows: The antibody-producing immunogen(having BSA as the carrier) obtained in Example 6 was converted into asolid phase on a microplate, and blood serum obtained from the immunizedanimal was added to the microplate to effect reaction. After washing,peroxidase (POD)-labeled anti-mouse IgG antibody was added to themicroplate to effect reaction. After washing, a color-producing solutioncontaining hydrogen peroxide and2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was addedto the microplate to develop color. Then, the antibody titer wasdetermined by measuring the absorbance at 415 nm on an EIA plate reader(manufactured by Bio-Rad).

After a total of five booster injections were given, it was noted thatthe antibody titer reached a plateau. Accordingly, exsanguination wasperformed and blood serum was separated to obtain 1.2 ml of antiserum.

This antiserum was centrifuged at 10,000 r.p.m. for 30 minutes to removeany insoluble matter, and then salted-out at 20° C. by adding 0.18 g ofsodium sulfate per ml of antiserum.

The resulting precipitate fraction of immunoglobulin was dissolved inthe least possible amount of 17.5 mM sodium dihydrogenphosphate-disodium hydrogen phosphate buffer (pH 6.3) and then dialyzedthoroughly against this 17.5 mM sodium dihydrogen phosphate-disodiumhydrogen phosphate buffer (pH 6.3).

After completion of the dialysis, the dialyzed solution was passedthrough a DEAE-cellulose ion exchange column (manufactured by Serva)which had been equilibrated with 17.5 mM sodium dihydrogenphosphate-disodium hydrogen phosphate buffer (pH 6.3). By collecting theflow-through fractions, there was obtained a mouse polyclonal antibodyto lipoprotein (a) specifically recognizing the amino acid sequencerepresented by SEQ ID NO:1 in the List of Sequences.

The amount of the antibody thus obtained was 1.0 mg as expressed interms of protein.

Reference Example 1

Reactivity of the polyclonal antibody to lipoprotein (a) obtained inExample 9 with the peptide represented by SEQ ID NO:8 in the List ofSequences

The reactivity of the polyclonal antibody to lipoprotein (a) obtained inExample 9 with the peptide represented by SEQ ID NO:8 in the List ofSequences, which is a peptide including the amino acid sequencerepresented by SEQ ID NO:i in the List of Sequences, was confirmed byELISA.

(1) The antibody-producing immunogen (having BSA as the carrier)obtained in Example 6 was dissolved in physiological saline (a 0.9%aqueous solution of sodium chloride) so as to give a concentration of 5μg/ml. This solution was added to wells of a 96-well microplate(manufactured by Nunc) in an amount of 100 μl per well and allowed tostand at 37° C. for 2 hours to convert the peptide into a solid phase.

(2) After this microplate was washed with a washing solutionphosphate-buffered physiological saline 5.59 mM disodium hydrogenphosphate, 1.47 mM potassium dihydrogen phosphate, 137 mM sodiumchloride, 2.68 mM potassium chloride (pH 7.2)! containing 0.05% Tween20!, 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphatebuffer (pH 7.2) containing 1% BSA was added to the wells thereof in anamount of 300 μl per well and allowed to stand at 37° C. for 2 hours toeffect blocking. Thereafter, the microplate was washed again with thewashing solution.

(3) The polyclonal antibody to lipoprotein (a) obtained in Example 9 wasdissolved in phosphate-buffered physiological saline containing 3% BSAso as to give a concentration of 60 μg/ml, and then serial-diluted 2- to2,048-fold with phosphate-buffered physiological saline containing 3%BSA to prepare a series of dilutions. These dilutions were separatelyadded to the wells of the microplate in an amount of 100 μl per well andallowed to stand at 37° C. for 2 hours to effect reaction. Thereafter,the microplate was washed with the washing solution.

(4) As a control, blood serum obtained from an unimmunized mouse wasdiluted 200-fold with phosphate-buffered physiological saline containing3% BSA, and then serial-diluted 2- to 2,048-fold with phosphate-bufferedphysiological saline containing 3% BSA to prepare a series of dilutions.These dilutions were separately added to the wells of another microplateas obtained from step (2) in an amount of 100 μl per well and allowed tostand at 37° C. for 2 hours to effect reaction. Thereafter, themicroplate was washed with the washing solution.

(5) Peroxidase (POD)-labeled anti-mouse IgG antibody (manufactured byAmersham) was diluted 2,000-fold with phosphate-buffered physiologicalsaline containing 3% BSA, added to the wells of the microplates obtainedfrom steps (3) and (4) in an amount of 100 μl per well, and allowed tostand at 37° C. for 2 hours to effect reaction.

(6) After these microplates were washed with the washing solution, aperoxidase reaction solution (prepared by adding 2 μl of 1.7% hydrogenperoxide to 1 ml of 50 mM disodium hydrogen phosphate-24 mM citric acidbuffer containing 3 mM o-phenylenediamine, immediately before use) wasadded to the wells thereof in an amount of 100 μl per well and reactedat room temperature. After 15 minutes, 6N sulfuric acid was added in anamount of 50 μl per well to stop the reaction.

(7) The absorbances at 492 nm of the wells of these microplates weremeasured on an EIA plate reader (manufactured by Bio-Rad).

The results of these measurements are shown in FIG. 11.

From these results, it has been confirmed that the polyclonal antibodyto lipoprotein (a) obtained in Example 9 specifically recognizes andcombines with the peptide represented by SEQ ID NO:8 in the List ofSequences which is a peptide including the amino acid sequencerepresented by SEQ ID NO:1 in the List of Sequences.

EXAMPLE 10

Preparation of a mouse polyclonal antibody to lipoprotein (a)specifically recognizing the amino acid sequence represented by SEQ IDNO:2 in the List of Sequences

The preparation of an antibody was carried out in the same manner as inExample 9, except that the antibody-producing immunogen (having KLH asthe carrier) obtained in Example 7 was dissolved in physiological salineso as to give a concentration of 500 μg/ml at the time of the initialimmunization, the same immunogen was dissolved in physiological salineso as to give a concentration of 250 μg/ml at the time of each boosterinjection, and the antibody-producing immunogen (having BSA as thecarrier) obtained in Example 7 was converted into a solid phase and usedto determine the antibody titer. Thus, there was obtained a mousepolyclonal antibody to lipoprotein (a) specifically recognizing theamino acid sequence represented by SEQ ID NO:2 in the List of Sequences.

The amount of the antibody thus obtained was 1.1 mg as expressed interms of protein.

Reference Example 2

Reactivity of the polyclonal antibody to lipoprotein (a) obtained inExample 10 with the peptide represented by SEQ ID NO:9 in the List ofSequences

The reactivity of the polyclonal antibody to lipoprotein (a) obtained inExample 10 with the peptide represented by SEQ ID NO:9 in the List ofSequences, which is a peptide including the amino acid sequencerepresented by SEQ ID NO:2 in the List of Sequences, was confirmed byELISA.

Measurements were made in the same manner as in Reference Example 1,except that the polyclonal antibody to lipoprotein (a) obtained inExample 10 was used at the same concentration in place of the polyclonalantibody to lipoprotein (a) obtained in Example 9, and theantibody-producing immunogen (having BSA as the carrier) obtained inExample 7 was used at the same concentration and converted into a solidphase on a microplate in place of the antibody-producing immunogenobtained in Example 6.

The results of these measurements are shown in FIG. 12.

From these results, it has been confirmed that the polyclonal antibodyto lipoprotein (a) obtained in Example 10 specifically recognizes andcombines with the peptide represented by SEQ ID NO:9 in the List ofSequences which is a peptide including the amino acid sequencerepresented by SEQ ID NO:2 in the List of Sequences.

Reference Example 3

Reactivity with lipoprotein (a) and LDL of the polyclonal antibodies tolipoprotein (a) obtained in Examples 9 and 10

The reactivity with lipoprotein (a) and LDL of each of the polyclonalantibodies to lipoprotein (a) obtained in Examples 9 and 10 wasconfirmed by the western blot technique.

(1) Human blood serum having a high lipoprotein (a) concentration wasultracentrifuged to separate a fraction having a specific gravity rangeof 1.05 to 1.12. Then, this serum fraction was subjected tolysine-Sepharose 4B affinity chromatography (manufactured byPharmacia-LKB). Thus, there was obtained purified lipoprotein (a).

(2) Human blood serum having a high LDL concentration wasultracentrifuged to separate a fraction having a specific gravity rangeof 1.006 to 1.063. Then, this serum fraction was subjected to affinitychromatography using anti-lipoprotein (a) antibody (manufactured byImmuno) as the ligand. The flow-through fractions were collected toobtain purified LDL.

(3) These lipoprotein (a) and LDL were each dissolved in physiologicalsaline (a 0.9% aqueous solution of sodium chloride) so as to give aconcentration of 0.5 mg/ml. 2 μl samples of these solutions weresubjected to electrophoresis using a Titan Gel LipoproteinElectrophoresis Kit (manufactured by Helena Laboratory). The supportingmedium was agarose gel. Using a barbital buffer (pH 8.8) as theelectrophoresis buffer, a voltage of 90 V was applied for 75 minutes.

(4) Using a Nova Blot Electrophoretic Transfer Kit (manufactured byPharmacia-LKB), transfer was carried out on a dry basis according to itsinstruction manual.

(5) The agarose gel obtained from step (3) was placed on a transferapparatus and a 9 cm×9 cm nitrocellulose membrane (manufactured byBio-Rad) was laid thereon. Using a transfer buffer comprising 48 mMTris, 39 mM glycine, 0.0375% (W/V) sodium dodecyl sulfate (SDS) and 20%(V/V) methanol, transfer was carried out by passing an electric currentof 65 mA for 2 hours.

(6) The nitrocellulose membrane having undergone the transfer was soakedovernight in 20 ml of phosphate-buffered physiological saline 5.59 mMdisodium hydrogen phosphate, 1.47 mM potassium dihydrogen phosphate, 137mM sodium chloride, 2.68 mM potassium chloride (pH 7.2)! containing 1%BSA at 4° C. to effect blocking.

(7) Then, this nitrocellulose membrane was washed by shaking it in 20 mlof a washing solution (phosphate-buffered physiological salinecontaining 0.05% Tween 20) for 10 minutes. This procedure was repeatedthree times.

(8) 80 μg each of the polyclonal antibodies to lipoprotein (a) obtainedin Examples 9 and 10 was dissolved in 20 ml of phosphate-bufferedphysiological saline. Two nitrocellulose membranes as obtained from step(7) were separately soaked in these two solutions at room temperaturefor 2 hours to effect reaction.

(9) As a control, the procedure described above in step (8) was repeatedby using a mixture of sheep anti-lipoprotein (a) antibody (manufacturedby Immuno) and goat anti-apolipoprotein B antibody to apolipoproteinB-100 being a component of LDL (manufactured by International Enzyme),both dissolved at the same concentration, in place of the polyclonalantibody to lipoprotein (a) obtained in Examples 9 and 10.

In addition, another nitrocellulose membrane as obtained from step (7),which was not acted on by any of the polyclonal antibodies obtained inExamples 9 and 10, sheep anti-lipoprotein (a) antibody and goatanti-apolipoprotein B antibody, was provided as a negative control.

(10) The nitrocellulose membranes subjected to the procedure describedabove in step (8) or (9) were washed by shaking them in 20 ml of thewashing solution for 10 minutes. This procedure was repeated threetimes.

(11) Next, peroxidase-labeled anti-mouse IgG antibody (manufacture byDako), peroxidase-labeled anti-sheep IgG antibody (manufactured by Dako)and peroxidase-labeled anti-goat IgG antibody (manufactured by Dako)were diluted 500-fold with phosphate-buffered physiological salinecontaining 3% BSA to prepare 20 ml of a solution, and the nitrocellulosemembranes were soaked therein at room temperature for 2 hours to effectreaction.

(12) These nitrocellulose membranes were washed by shaking them in 20 mlof the washing solution for 10 minutes. This procedure was repeatedthree times.

(13) The nitrocellulose membranes obtained from step (12) were soaked in20 ml of phosphate-buffered physiological saline containing 0.025%3,3'-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide atroom temperature for 15 minutes to develop color.

The results of this western blot technique are shown in FIG. 13.

In FIG. 13, P represents the control, N represents the negative control,and numerals 1 and 2 represent the nitrocellulose membranes acted on bythe polyclonal antibodies to lipoprotein (a) obtained in Examples 9 and10, respectively. In each nitrocellulose membrane, lipoprotein (a) istransferred to the left-hand part and LDL to the right-hand part.

As can be seen from these results, the polyclonal antibodies tolipoprotein (a) obtained in Examples 9 and 10 exhibit color developmentat the same position as the commercially available anti-lipoprotein (a)antibody does, but no color development at the position where thecommercially available anti-apolipoprotein B antibody exhibits colordevelopment. Thus, it has been confirmed that these polyclonalantibodies combine specifically with lipoprotein (a) and do not combinewith LDL.

Moreover, no color development is observed in the negative control whichwas not acted on by any of the polyclonal antibodies obtained inExamples 9 and 10, the commercially available anti-lipoprotein (a)antibody and the commercially available anti-apolipoprotein B antibody,indicating that nonspecific color development did not take place.

Reference Example 4

Reactivity with plasminogen of the polyclonal antibodies to lipoprotein(a) obtained in Examples 9 and 10

The reactivity with plasminogen of each of the polyclonal antibodies tolipoprotein (a) obtained in Examples 9 and 10 was confirmed by thewestern blot technique.

(1) Human blood plasma having a high plasminogen concentration wasultracentrifuged to separate a fraction having a specific gravity of1.21 or greater. This plasma fraction was subjected to lysine-Sepharose4B affinity chromatography (manufactured by Pharmacia-LKB) and then toaffinity chromatography using anti-lipoprotein (a) antibody(manufactured by Immuno) as the ligand. The flow-through fractions werecollected to obtain purified plasminogen.

(2) This plasminogen was dissolved in physiological saline (a 0.9%aqueous solution of sodium chloride) so as to give a concentration of1.0 mg/ml, and a 10 μl sample of this solution was subjected toelectrophoresis. The supporting medium was 3-12% SDS polyacrylamide gel.Using 25 mM Tris-0.19M glycine buffer containing 0.1% SDS as theelectrophoresis buffer, an electric current of 20 mA was passed for 120minutes.

(3) Using a Nova Blot Electrophoretic Transfer Kit (manufactured byPharmacia-LKB), transfer was carried out on a dry basis according to itsinstruction manual.

(4) The 3-12% SDS polyacrylamide gel obtained from step (2) was placedon a transfer apparatus and a 9 cm×9 cm nitrocellulose membrane(manufactured by Bio-Rad) was laid thereon. Using a transfer buffercomprising 48 mM Tris, 39 mM glycine, 0.0375% (W/V) sodium dodecylsulfate (SDS) and 20% (V/V) methanol, transfer was carried out bypassing an electric current of 65 mA for 2 hours.

The succeeding procedures were substantially the same as those describedin and after step (6) of Reference Example 3. However, it is to beunderstood that, although a mixture of sheep anti-lipoprotein (a)antibody and goat anti-apolipoprotein B antibody was used as a controlin step (9) of Reference Example 3, goat anti-plasminogen antibody(manufactured by Medical Biological Laboratory) was used at the sameconcentration in this reference example.

The results of this western blot technique are shown in FIG. 14.

In FIG. 14, P represents the control, N represents the negative control,and numerals 1 and 2 represent the nitrocellulose membranes acted on bythe polyclonal antibodies to lipoprotein (a) obtained in Examples 9 and10, respectively.

As can be seen from these results, the polyclonal antibodies tolipoprotein (a) obtained in Examples 9 and 10 exhibit no colordevelopment at the position where the commercially availableanti-plasminogen B antibody exhibits color development. Thus, it hasbeen confirmed that these polyclonal antibodies do not combine withplasminogen.

Moreover, no color development is observed in the negative control whichwas not acted on by the polyclonal antibodies obtained in Examples 9 and10 or the like, indicating that nonspecific color development did nottake place.

EXAMPLE 11

Preparation of a mouse polyclonal antibody to apolipoprotein (a)specifically recognizing the amino acid sequence represented by SEQ IDNO:3 in the List of Sequences

The preparation of an antibody was carried out in the same manner as inExample 9, except that the antibody-producing immunogen (having KLH asthe carrier) obtained in Example 8 was dissolved in physiological salineso as to give a concentration of 400 μg/ml at the time of the initialimmunization, the same immunogen was dissolved in physiological salineso as to give a concentration of 200 μg/ml at the time of each boosterinjection, and the antibody-producing immunogen (having BSA as thecarrier) obtained in Example 8 was converted into a solid phase and usedto determine the antibody titer. Thus, there was obtained a mousepolyclonal antibody to apolipoprotein (a) specifically recognizing theamino acid sequence represented by SEQ ID NO:3 in the List of Sequences.

The amount of the antibody thus obtained was 1.0 mg as expressed interms of protein.

Reference Example 5

Reactivity of the polyclonal antibody to apolipoprotein (a) obtained inExample 11 with the peptide represented by SEQ ID NO:10 in the List ofSequences

The reactivity of the polyclonal antibody to apolipoprotein (a) obtainedin Example 11 with the peptide represented by SEQ ID NO:10 in the Listof Sequences, which is a peptide including the amino acid sequencerepresented by SEQ ID NO:3 in the List of Sequences, was confirmed byELISA.

Measurements were made in the same manner as in Reference Example 1,except that the polyclonal antibody to apolipoprotein (a) obtained inExample 11 was used at the same concentration in place of the polyclonalantibody to lipoprotein (a) obtained in Example 9, and theantibody-producing immunogen (having BSA as the carrier) obtained inExample 8 was used at the same concentration and converted into a solidphase on a microplate in place of the antibody-producing immunogenobtained in Example 6.

The results of these measurements are shown in FIG. 15.

From these results, it has been confirmed that the polyclonal antibodyto apolipoprotein (a) obtained in Example 11 specifically recognizes andcombines with the peptide represented by SEQ ID NO:10 in the List ofSequences which is a peptide including the amino acid sequencerepresented by SEQ ID NO:3 in the List of Sequences.

Reference Example 6

Reactivity with apolipoprotein (a) of the polyclonal antibody toapolipoprotein (a) obtained in Example 11

The reactivity with apolipoprotein (a) of the polyclonal antibody toapolipoprotein (a) obtained in Example 11 was confirmed by the westernblot technique.

(1) Four types of human blood sera having a high lipoprotein (a)concentration were ultracentrifuged to separate a fraction having aspecific gravity range of 1.05 to 1.12. Then, the resulting serumfractions were subjected to lysine-Sepharose 4B affinity chromatography(manufactured by Pharmacia-LKB). Thus, there were obtained four types ofpurified lipoprotein (a).

Then, these four types of purified lipoprotein (a) were treated by theaddition of 1 mM dithiothreitol and ultracentrifuged again to separate afraction having a specific gravity of 1.21 or greater. Thus, thelow-density lipoprotein (LDL) moiety was removed to obtain four types ofpurified apolipoprotein (a).

(2) These four types of purified apolipoprotein (a) were each dissolvedin a sample buffer 10 mM Tris, 1% sodium dodecyl sulfate (SDS) (pH6.80)! so as to give a concentration of 0.5 mg/ml. 2 μl samples of thesesolutions were subjected to SDS polyacrylamide gel electrophoresis using4% SDS polyacrylamide gel (manufactured by Tefco). Using a buffer (pH8.64) comprising 40 mM Tris, 40 mM boric acid and 0.1% sodium dodecylsulfate (SDS) as the cathode-side buffer and 0.43M Tris buffer (pH 9.18)as the anode-side buffer, an electric current of 20 mA was passed for 75minutes.

(3) Using a Nova Blot Electrophoretic Transfer Kit (manufactured byPharmacia-LKB), transfer was carried out on a dry basis according to itsinstruction manual.

(4) The 4% SDS polyacrylamide gel obtained from step (2) was placed on atransfer apparatus and a 9 cm×9 cm nitrocellulose membrane (manufacturedby Bio-Rad) was laid thereon. Using a transfer buffer comprising 48 mMTris, 39 mM glycine, O.0375% (W/V) SDS and 20% (V/V) methanol, transferwas carried out by passing an electric current of 65 mA for 2 hours.

(5) The nitrocellulose membrane having undergone the transfer was soakedovernight in 20 ml of phosphate-buffered physiological saline 5.59 mMdisodium hydrogen phosphate, 1.47 mM potassium dihydrogen phosphate, 137mM sodium chloride, 2.68 mM potassium chloride (pH 7.2)! containing 1%BSA at 4° C. to effect blocking.

(6) Then, this nitrocellulose membrane was washed by shaking it in 20 mlof a washing solution (phosphate-buffered physiological salinecontaining 0.05% Tween 20) for 10 minutes. This procedure was repeatedthree times.

(7) 80 μg of the polyclonal antibody to apolipoprotein (a) obtained inExample 11 was dissolved in 20 ml of phosphate-buffered physiologicalsaline. The nitrocellulose membrane subjected to the procedure describedin step (6) was soaked in this solution at room temperature for 2 hoursto effect reaction.

(8) As a control, the procedure described above in step (7) was repeatedby using sheep anti-lipoprotein (a) antibody (manufactured by Immuno)reacting with apolipoprotein (a), dissolved at the same concentration,in place of the polyclonal antibody to apolipoprotein (a) obtained inExamples 11. In addition, another nitrocellulose membrane as obtainedfrom step (6), which was not acted on by either of the polyclonalantibody to apolipoprotein (a) obtained in Examples 11 and sheepanti-lipoprotein (a) antibody, was provided as a negative control.

(9) The nitrocellulose membranes subjected to the procedure describedabove in step (7) or (8) were washed by shaking them in 20 ml of thewashing solution for 10 minutes. This procedure was repeated threetimes.

(10) Next, peroxidase-labeled anti-mouse IgG antibody (manufacture byDako) and peroxidase-labeled anti-sheep IgG antibody (manufactured byDako) were diluted 500-fold with phosphate-buffered physiological salinecontaining 3% BSA to prepare 20 ml of a solution, and the nitrocellulosemembranes were soaked therein at room temperature for 2 hours to effectreaction.

(11) These nitrocellulose membranes were washed by shaking them in 20 mlof the washing solution for 10 minutes. This procedure was repeatedthree times.

(12) The nitrocellulose membranes obtained from step (11) were soaked in20 ml of phosphate-buffered physiological saline containing 0.025%3,3'-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide atroom temperature for 15 minutes to develop color.

(13) In order to set up a reference standard for identifying the bandsobtained in this western blot technique, the same procedures asdescribed above were repeated using purified apolipoprotein B-100(prepared domestically), goat anti-apolipoprotein B antibody(manufactured by International Enzyme) and peroxidase-labeled anti-goatIgG antibody (manufactured by Dako). Thus, there was obtained a band ofapolipoprotein B-100.

The results of this western blot technique are shown in FIGS. 16, 17 and18.

FIG. 16 shows the nitrocellulose membrane acted on by the polyclonalantibody to apolipoprotein (a) obtained in Example 11.

In FIG. 16, numerals 1 to 4 represent the respective electrophoreticpatterns of the four types of purified apolipoprotein (a) derived fromhuman blood serum, and B shows the position of the band ofapolipoprotein B-100 useful as a reference standard for identifying theresulting electrophoretic bands.

On the basis of positional relationship with this band of apolipoproteinB-100 G. Utermann et al., J. Clin. Invest., 80, 458-465(1987)!, it canbe seen that the bands observed in pattern 1 represent F and B isotypesof apolipoprotein (a), those observed in pattern 2 represent S4 and Fisotypes, those observed in pattern 3 represent S4 and S3 isotypes, andthose observed in pattern 4 represent S4 and S2 isotypes.

FIG. 17 shows the negative control in which numerals 1 to 4 representthe respective electrophoretic patterns of the four types of purifiedapolipoprotein (a) derived from human blood serum.

FIG. 18 shows the control in which the nitrocellulose membrane was actedon by anti-lipoprotein (a) antibody reacting with apolipoprotein (a).The sample used was the purified apolipoprotein (a) derived from bloodserum which corresponds to the above-described pattern 1. B shows theposition of the band of apolipoprotein B-100 as a reference standard.

As can be seen from FIGS. 16 and 18, the polyclonal antibody toapolipoprotein (a) obtained in Example 11 exhibits color development atthe same position as anti-lipoprotein (a) antibody reacting withapolipoprotein (a) does. Thus, it has been confirmed that thispolyclonal antibody combines specifically with apolipoprotein (a).

Moreover, in FIG. 16, it has been confirmed that the polyclonal antibodyto apolipoprotein (a) obtained in Example 11 react with various isotypesof apolipoprotein (a).

Furthermore, in FIG. 17, no color development is observed in thenegative control which was not acted on by the polyclonal antibody toapolipoprotein (a) obtained in Example 11 or anti-lipoprotein (a)antibody, indicating that nonspecific color development did not takeplace.

Reference Example 7

Reactivity with lipoprotein (a) of the polyclonal antibody toapolipoprotein (a) obtained in Example 11

The reactivity with lipoprotein (a) of the polyclonal antibody toapolipoprotein (a) obtained in Example 11 was confirmed by the westernblot technique.

(1) Human blood serum having a high lipoprotein (a) concentration wasultracentrifuged to separate a fraction having a specific gravity rangeof 1.05 to 1.12. This serum fraction was subjected to lysine-Sepharose4B affinity chromatography (manufactured by Pharmacia-LKB). Thus, therewas obtained purified lipoprotein (a).

(2) This purified lipoprotein (a) was dissolved in physiological saline(a 0.9% aqueous solution of sodium chloride) so as to give aconcentration of 0.5 mg/ml, and a 2 μl sample of this solution wassubjected to electrophoresis using a Titan Gel LipoproteinElectrophoresis Kit (manufactured by Helena Laboratory). The supportingmedium was agarose gel. Using a barbital buffer (pH 8.8) as theelectrophoresis buffer, a voltage of 90 V was applied for 75 minutes.

(3) Using a Nova Blot Electrophoretic Transfer Kit (manufactured byPharmacia-LKB), transfer was carried out on a dry basis according to itsinstruction manual.

(4) The agarose gel obtained from step (2) was placed on a transferapparatus and a 9 cm×9 cm nitrocellulose membrane (manufactured byBio-Rad) was laid thereon. Using a transfer buffer comprising 48 mMTris, 39 mM glycine, 0.0375% (W/V) SDS and 20% (V/V) methanol, transferwas carried out by passing an electric current of 65 mA for 2 hours.

The succeeding procedures were substantially the same as those describedin steps (5) to (12) of Reference Example 6.

The results of this western blot technique are shown in FIG. 19.

In FIG. 19, P represents the control, N represents the negative control,and S represents the nitrocellulose membrane acted on by the polyclonalantibody to apolipoprotein (a) obtained in Example 11.

By comparison between S, and the control and the negative control inFIG. 19, it has been confirmed that the polyclonal antibody toapolipoprotein (a) obtained in Example 11 does not react withlipoprotein (a).

Reference Example 8

Reactivity with plasminogen of the polyclonal antibody to apolipoprotein(a) obtained in Example 11

The reactivity with plasminogen of the polyclonal antibody toapolipoprotein (a) obtained in Example 11 was confirmed by the westernblot technique.

The western blot technique was carried out in the same manner as inReference Example 4, except that the polyclonal antibody toapolipoprotein (a) obtained in Example 11 was used in place of thepolyclonal antibodies to lipoprotein (a) obtained in Examples 9 and 10.

As a result, the polyclonal antibody to apolipoprotein (a) obtained inExample 11 exhibits no color development at the position where thecommercially available anti-plasminogen antibody exhibits colordevelopment. Thus, it has been confirmed that this polyclonal antibodydoes not combine with plasminogen.

Moreover, no color development is observed in the negative control whichwas not acted on by the polyclonal antibody to apolipoprotein (a)obtained in Example 11 or the like, indicating that nonspecific colordevelopment did not take place.

EXAMPLE 12

Preparation of a rabbit polyclonal antibody to lipoprotein (a)specifically recognizing the amino acid sequence represented by SEQ IDNO:i in the List of Sequences

The antibody-producing immunogen (having KLH as the carrier) obtained inExample 6 was dissolved in physiological saline (a 0.9% aqueous solutionof sodium chloride) so as to give a concentration of 1.6 mg/ml. Thissolution was mixed with an equal amount of Freund's complete adjuvant toform an emulsion, and 1 ml of this emulsion was subcutaneously injectedat 20 or more sites of the back of a rabbit (Japanese White) aged 3months for purposes of immunization.

Two weeks after the initial immunization, the above-describedantibody-producing immunogen was dissolved in physiological saline so asto give a concentration of 0.8 mg/ml. This solution was mixed with anequal amount of Freund's incomplete adjuvant to form an emulsion, and 1ml of this emulsion was injected as a booster. This booster injectionwas repeated at intervals of 2 weeks.

The antibody titer in the blood serum of this rabbit, which was animmunized animal, was measured by an enzyme immunoassay (ELISA, EIA) atintervals of one week. This ELISA was carried out as follows: Theantibody-producing immunogen (having BSA as the carrier) obtained inExample 6 was converted into a solid phase on a microplate, and bloodserum obtained from the immunized animal was added thereto to effectreaction. After washing, peroxidase (POD)-labeled anti-mouse IgGantibody was added to the microplate to effect reaction. After washing,a color-producing solution containing hydrogen peroxide and2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was addedto the microplate to develop color. Then, the antibody titer wasdetermined by measuring the absorbance at 415 nm on an EIA plate reader(manufactured by Bio-Rad).

After a total of five booster injections were given, it was noted thatthe antibody titer reached a plateau. Accordingly, exsanguination wasperformed and blood serum was separated to obtain 82 ml of antiserum.

This antiserum was centrifuged at 10,000 r.p.m. for 30 minutes to removeany insoluble matter, and then salted-out at 20° C. by adding 0.18 g ofsodium sulfate per ml of antiserum. The resulting precipitate fractionof immunoglobulin was dissolved in the least possible amount of 17.5 mMsodium dihydrogen phosphate-disodium hydrogen phosphate buffer (pH 6.3)and then dialyzed thoroughly against this 17.5 mM sodium dihydrogenphosphate-disodium hydrogen phosphate buffer (pH 6.3).

After completion of the dialysis, the dialyzed solution was passedthrough a DEAE-cellulose ion exchange column (manufactured by Serva)which had been equilibrated with 17.5 mM sodium dihydrogenphosphate-disodium hydrogen phosphate buffer (pH 6.3). By collecting theflow-through fractions, there was obtained a rabbit polyclonal antibodyto lipoprotein (a) specifically recognizing the amino acid sequencerepresented by SEQ ID NO:i in the List of Sequences.

The amount of the antibody thus obtained was 0.74 g as expressed interms of protein.

EXAMPLE 13

Preparation of a rabbit polyclonal antibody to lipoprotein (a)specifically recognizing the amino acid sequence represented by SEQ IDNO:2 in the List of Sequences

The preparation of an antibody was carried out in the same manner as inExample 12, except that the antibody-producing immunogen (having KLH asthe carrier) obtained in Example 7 was dissolved in physiological salineso as to give a concentration of 2.2 mg/ml at the time of the initialimmunization, the same immunogen was dissolved in physiological salineso as to give a concentration of 1.1 mg/ml at the time of each boosterinjection, and the antibody-producing immunogen (having BSA as thecarrier) obtained in Example 7 was converted into a solid phase and usedto determine the antibody titer. Thus, there was obtained a rabbitpolyclonal antibody to lipoprotein (a) specifically recognizing theamino acid sequence represented by SEQ ID NO:2 in the List of Sequences.

The amount of the antibody thus obtained was 0.78 g as expressed interms of protein.

EXAMPLE 14

Preparation of a rabbit polyclonal antibody to apolipoprotein (a)specifically recognizing the amino acid sequence represented by SEQ IDNO:3 in the List of Sequences

The preparation of an antibody was carried out in the same manner as inExample 12, except that the antibody-producing immunogen (having KLH asthe carrier) obtained in Example 8 was dissolved in physiological salineso as to give a concentration of 1.7 mg/ml at the time of the initialimmunization, the same immunogen was dissolved in physiological salineso as to give a concentration of 0.9 mg/ml at the time of each boosterinjection, and the antibody-producing immunogen (having BSA as thecarrier) obtained in Example 8 was converted into a solid phase and usedto determine the antibody titer. Thus, there was obtained a rabbitpolyclonal antibody to apolipoprotein (a) specifically recognizing theamino acid sequence represented by SEQ ID NO:3 in the List of Sequences.

The amount of the antibody thus obtained was 0.71 g as expressed interms of protein.

EXAMPLE 15

Method for the determination of lipoprotein (a) by turbidimetricimmunoassay

A system for the determination of lipoprotein (a) by turbidimetricimmunoassay was established using the polyclonal antibody to lipoprotein(a) obtained in Example 9.

(1) For use in determination, 40 mM Tris-hydrochloric acid buffer (pH7.0) containing 300 mM sodium chloride and 5% (W/W) polyethylene glycol6000, and phosphate-buffered physiological saline 5.59 mM disodiumhydrogen phosphate, 1.47 mM potassium dihydrogen phosphate, 137 mMsodium chloride, 2.68 mM potassium chloride (pH 7.2)! containing 6 mg/mlof the polyclonal antibody to lipoprotein (a) obtained in Example 9 wereprepared as reagents 1 and 2, respectively.

(2) Blood serum having a lipoprotein (a) concentration of 77.0 mg/dl wasdiluted with physiological saline (a 0.9% aqueous solution of sodiumchloride) in three steps. Thus, three samples having lipoprotein (a)concentrations of 25.7 mg/dl, 51.3 mg/dl and 77.0 mg/dl were prepared.

(3) Measurements were made by using a COBAS MIRA automatic analyzermanufactured by F. Hoffmann La Roche. More specifically, 3 μl eachsample was mixed with 300 μl of reagent 1 and this mixture was warmed at37° C. for 5 minutes. After 20 μl of reagent 2 was added thereto andmixed therewith, the resulting mixture was reacted at 37° C. for 5minutes. Then, its absorbance at 340 nm was measured.

The calibration curve obtained by measuring the three samples is shownin FIG. 20.

As can be seen from this figure, the calibration curve used in thismethod is a straight line passing through the origin. Thus, it has beenconfirmed that the method for the determination of lipoprotein (a) inaccordance with the present invention enables quantitative determinationof lipoprotein (a).

EXAMPLE 16

Method for the determination of lipoprotein (a) by turbidimetricimmunoassay

A system for the determination of lipoprotein (a) by turbidimetricimmunoassay was established using the polyclonal antibody to lipoprotein(a) obtained in Example 10.

Measurements were made in the same manner as in Example 15, except that,as a component of reagent 2 prepared in step (1) of Example 15, thepolyclonal antibody to lipoprotein (a) obtained in Example 10 was usedat the same concentration in place of the polyclonal antibody tolipoprotein (a) obtained in Example 9.

The calibration curve obtained by measuring the three samples is shownin FIG. 21.

As can be seen from this figure, the calibration curve used in thismethod is a straight line passing through the origin. Thus, it has beenconfirmed that the method for the determination of lipoprotein (a) inaccordance with the present invention enables quantitative determinationof lipoprotein (a).

EXAMPLE 17

Method for the determination of lipoprotein (a) by ELISA

A system for the determination of lipoprotein (a) by ELISA wasestablished using the polyclonal antibody to lipoprotein (a) obtained inExample 9.

(1) A 50 μg/ml solution of anti-lipoprotein (a) antibody (manufacturedby International Enzyme) was added to wells of a 96-well microplate(manufactured by Nunc) in an amount of 100 μl per well and allowed tostand at 37° C. for 2 hours to convert the anti-lipoprotein (a) antibodyinto a solid phase (capture antibody).

(2) After this microplate was washed with a washing solutionphosphate-buffered physiological saline 5.59 mM disodium hydrogenphosphate, 1.47 mM potassium dihydrogen phosphate, 137 mM sodiumchloride, 2.68 mM potassium chloride (pH 7.2)! containing 0.05% Tween20!, 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphatebuffer (pH 7.2) containing 1% BSA was added to the wells thereof in anamount of 300 μl per well and allowed to stand at 37° C. for 2 hours toeffect blocking. Thereafter, the microplate was washed again with thewashing solution.

(3) Blood serum having a lipoprotein (a) concentration of 100 mg/dl wasdiluted with physiological saline (a 0.9% aqueous solution of sodiumchloride) in five steps. Thus, five samples having lipoprotein (a)concentrations of 20 mg/dl, 40 mg/dl, 60 mg/dl, 80 mg/dl and 100 mg/dlwere prepared.

(4) The above-described five samples were diluted 500-fold withphysiological saline. These dilutions were separately pipetted into thewells of the microplate obtained from step (2) in an amount of 100 μlper well and allowed to stand at 37° C. for 2 hours to effect anantigen-antibody reaction.

(5) After this microplate was washed with the washing solution, thepolyclonal antibody to lipoprotein (a) obtained in Example 9 wasdissolved in phosphate-buffered physiological saline containing 3% BSAso as to give a concentration of 50 μg/ml, and the resulting solutionwas added to the wells of the microplate in an amount of 100 μl per welland allowed to stand at 37° C. for 2 hours to effect reaction (primaryantibody). Thereafter, the microplate was washed with the washingsolution.

(6) Peroxidase (POD)-labeled anti-mouse IgG antibody (manufactured byAmersham) was diluted 2,000-fold with phosphate-buffered physiologicalsaline containing 3% BSA, added to the wells of the microplate obtainedfrom step (5) in an amount of 100 μl per well, and allowed to stand at37° C. for 2 hours to effect reaction (secondary antibody).

(7) After this microplate was washed with the washing solution, aperoxidase reaction solution (prepared by adding 2 μl of 1.7% hydrogenperoxide to 1 ml of 50 mM disodium hydrogen phosphate-24 mM citric acidbuffer containing 3 mM o-phenylenediamine, immediately before use) wasadded to the wells thereof in an amount of 100 μl per well and reactedat room temperature. After 15 minutes, 6N sulfuric acid was added to thewells thereof in an amount of 50 μl per well to stop the reaction.

(8) The absorbances at 492 nm of the wells of this microplate weremeasured on an EIA microplate reader (manufactured by Bio-Rad).

The calibration curve obtained by measuring the five samples is shown inFIG. 22.

From these results, it has been confirmed that the method for thedetermination of lipoprotein (a) in accordance with the presentinvention enables quantitative determination of lipoprotein (a).

EXAMPLE 18

Method for the determination of lipoprotein (a) by ELISA

A system for the determination of lipoprotein (a) by ELISA wasestablished using the polyclonal antibody to lipoprotein (a) obtained inExample 10.

Measurements were made in the same manner as Example 17, except that thepolyclonal antibody to lipoprotein (a) obtained in Example 10 was usedat the same concentration in step (5) of Example 17, in place of thepolyclonal antibody to lipoprotein (a) obtained in Example 9.

The calibration curve obtained by measuring the five samples is shown inFIG. 23.

From these results, it has been confirmed that the method for thedetermination of lipoprotein (a) in accordance with the presentinvention enables quantitative determination of lipoprotein (a).

EXAMPLE 19

Comparison of the measured values for lipoprotein (a)

The measured values obtained by the present method for the determinationof lipoprotein (a) by ELISA using the polyclonal antibody to lipoprotein(a) obtained in Example 10 were compared with those obtained by ELISAusing the lipoprotein (a)-determining reagent of company A and byturbidimetric immunoassay using the lipoprotein (a)-determining reagentof company B.

Samples 1, 2 and 3 comprising three types of blood sera were measuredaccording to the present method for the determination of lipoprotein (a)by ELISA using the polyclonal antibody to lipoprotein (a) obtained inExample 10. The ELISA was carried out in the same manner as in Example17.

The measurement of three samples by using the lipoprotein(a)-determining reagent of company A and the lipoprotein (a)-determiningreagent of company B was made according to their instruction manuals.

The results of these measurements are summarized in Table 4.

                  TABLE 4                                                         ______________________________________                                               Method of the                                                                            Reagent of                                                                              Reagent of                                               invention  company A company B                                         ______________________________________                                        Sample 1 58.0         51.3      54.0                                          Sample 2 0.0          0.0       5.0 or less                                   Sample 3 42.0         42.0      44.9                                          ______________________________________                                    

(The values are expressed in mg/dl.)

From these results, it has been confirmed that the measured values forlipoprotein (a) obtained by the present method for the determination oflipoprotein (a) are substantially the same as those obtained by themethods currently in use and, therefore, the method for thedetermination of lipoprotein (a) in accordance with the presentinvention can be practically used for purposes of clinical examination.

Reference Example 9

Influence of plasminogen on the present method for the determination oflipoprotein (a)

In order to investigate the influence of plasminogen on the presentmethod for the determination of lipoprotein (a), the followingexperiments were carried out using four types of primary antibodies(A-1, A-2, B and C).

(1) Goat anti-plasminogen antibody (manufactured by Medical BiologicalLaboratory) was converted into a solid phase on a microplate and used asa capture antibody.

(2) Four types of antibodies (A-1, A-2, B and C) were provided asprimary antibodies. A-1 comprised the polyclonal antibody to lipoprotein(a) obtained in Example 9, A-2 comprised the polyclonal antibody tolipoprotein (a) obtained in Example 10, B comprised a mouse monoclonalantibody combining with plasminogen but not combining with lipoprotein(a) (i.e., an antibody combining specifically with the Kringle 1,Kringle 2 and Kringle 3 portions of plasminogen) (prepareddomestically), and C comprised a mouse polyclonal antibody obtainedusing lipoprotein (a) as the immunogen but not subjected to anabsorption treatment with plasminogen (prepared domestically).

(3) Peroxidase-labeled anti-mouse IgG antibody (manufactured byAmersham) was provided as a secondary antibody.

(4) Using purified plasminogen (prepared domestically), five sampleshaving plasminogen concentrations of 44 mg/dl, 88 mg/dl, 132 mg/dl, 176mg/dl and 220 mg/dl were prepared. Prior to use, these samples werediluted 3,000-fold with physiological saline (a 0.9% aqueous solution ofsodium chloride) and used for measurements.

(5) The above-described five samples were measured by ELISA using thecapture antibody described above in step (1), the four primaryantibodies described above in step (2), and the secondary antibodydescribed above in step (3). The ELISA was carried out in the samemanner as in Example 17.

The results obtained by measuring the five plasminogen samples are shownin FIG. 24.

It can be seen from these results that plasminogen is measured togetherin the measuring system using, as the primary antibody, an antibody (B)combining solely with plasminogen and the measuring system using, as theprimary antibody, an antibody (C) obtained from an immunogen comprisinglipoprotein (a).

In contrast, plasminogen is by no means measured together in the presentsystem for the determination of lipoprotein (a) using, as the primaryantibody, the polyclonal antibody (A-1, A-2) to lipoprotein (a) obtainedin Example 9 or 10.

Thus, it has been confirmed that the method for the determination oflipoprotein (a) in accordance with the present invention is a methodcapable of determining the concentration of lipoprotein (a) accuratelywithout undergoing the influence of any plasminogen present in thesamples.

EXAMPLE 20

Method for the determination of apolipoprotein (a) by ELISA

A system for the determination of apolipoprotein (a) by ELISA wasestablished using the polyclonal antibody to apolipoprotein (a) obtainedin Example 11.

(1) Anti-lipoprotein (a) antibody also combining with apolipoprotein (a)(manufactured by International Enzyme) was treated with pepsin tofragment it to F(ab')₂. A 15 μg/ml solution of this antibody fragmentwas added to wells of a 96-well microplate (manufactured by Nunc) in anamount of 100 μl per well and allowed to stand at 37° C. for 2 hours toconvert the anti-lipoprotein (a) antibody fragment into a solid phase(capture antibody).

(2) After this microplate was washed with a washing solutionphosphate-buffered physiological saline 5.59 mM disodium hydrogenphosphate, 1.47 mM potassium dihydrogen phosphate, 137 mM sodiumchloride, 2.68 mM potassium chloride (pH 7.2)! containing 0.05% Tween20!, 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphatebuffer (pH 7.2) containing 1% BSA was added to the wells thereof in anamount of 300 μl per well and allowed to stand at 37° C. for 2 hours toeffect blocking. Thereafter, the microplate was washed again with thewashing solution.

(3) Human blood serum having a high lipoprotein (a) concentration wasultracentrifuged to separate a fraction having a specific gravity rangeof 1.05 to 1.12. Then, the resulting serum fraction was subjected tolysine-Sepharose 4B affinity chromatography (manufactured byPharmacia-LKB). Thus, there was obtained purified lipoprotein (a).

Then, this purified lipoprotein (a) was treated by the addition of 1 mMdithiothreitol and ultracentrifuged again to separate a fraction havinga specific gravity of 1.21 or greater. Thus, the LDL moiety was removedto obtain purified apolipoprotein (a).

This purified apolipoprotein (a) was diluted with physiological saline(a 0.9% aqueous solution of sodium chloride) so as to give aconcentration of 40 mg/dl and then diluted with physiological saline infour steps. Thus, four samples having apolipoprotein (a) concentrationsof 10 mg/dl, 20 mg/dl, 30 mg/dl and 40 mg/dl were prepared.

(4) The above-described four samples were diluted 100-fold with a samplediluting solution 10 mM Tris, 0.9% sodium chloride, 1% BSA(pH 8.0)!.These dilutions were separately pipetted into the wells of themicroplate obtained from step (2) in an amount of 100 μl per well andallowed to stand at 37° C. for 2 hours to effect an antigen-antibodyreaction.

(5) After this microplate was washed with the washing solution, thepolyclonal antibody to apolipoprotein (a) obtained in Example 11 wasdissolved in phosphate-buffered physiological saline containing 3% BSAso as to give a concentration of 50 μg/ml, and the resulting solutionwas added to the wells of the microplate in an amount of 100 μl per welland allowed to stand at 37° C. for 2 hours to effect reaction (primaryantibody). Thereafter, the microplate was washed with the washingsolution.

The succeeding procedures were substantially the same as those describedin steps (6) to (8) of Example 17.

The calibration curve obtained by measuring the four samples is shown inFIG. 25.

From these results, it has been confirmed that the method for thedetermination of apolipoprotein (a) in accordance with the presentinvention enables quantitative determination of apolipoprotein (a).

EXAMPLE 21

Influence of serum samples on the method for the determination ofapolipoprotein (a)

In the method for the determination of apolipoprotein (a) by ELISA usingthe polyclonal antibody to apolipoprotein (a) obtained in Example 11, itwas confirmed by addition and recovery tests that the method was notinfluenced by serum samples.

(1) Three types of blood sera (A, B, C) were provided and the followingsamples were prepared using them as base materials.

(i) Three samples prepared by mixing 0.1 ml of physiological saline (a0.9% aqueous solution of sodium chloride) with 0.9 ml of each of thethree blood sera (A, B, C).

(ii) Three samples prepared by diluting the purified apolipoprotein (a)obtained in Example 20 with physiological saline so as to give aconcentration of 100 mg/dl and mixing 0.1 ml of this solution with 0.9ml of each of the three blood sera (A, B, C) to increase theapolipoprotein (a) concentrations of the three serum samples prepared in(i) by 10 mg/ml.

(iii) A sample prepared by diluting the purified apolipoprotein (a)obtained in Example 20 with physiological saline so as to give aconcentration of 10 mg/dl.

(2) The absorbances of the above-described seven samples were measuredaccording to the method for the determination of apolipoprotein (a) byELISA as described in Example 20. The results thus obtained are shown inTable 5.

                  TABLE 5                                                         ______________________________________                                                        Serum A                                                                              Serum B  Serum C                                       ______________________________________                                        (i) Absorbance of each                                                                          0.047    0.060    0.011                                     sample comprising a                                                           blood serum mixed with                                                        physiological saline                                                          (iii) Absorbance of                                                                             0.110    0.110    0.110                                     10 mg/dl of purified                                                          apolipoprotein (a)                                                            (iv) Theoretical absorb-                                                                        0.157    0.170    0.121                                     ance of each serum                                                            sample of (i) having                                                          an apolipoprotein (a)                                                         concentration                                                                 increased by 10 mg/dl                                                          (i) + (iii)!                                                                 (ii) Measured absorbance                                                                        0.165    0.167    0.132                                     of each serum sample                                                          of (i) having an apo-                                                         lipoprotein (a) concen-                                                       tration increased by                                                          10 mg/dl                                                                      (v) Percentage of the                                                                           105%     98.2%    109%                                      measured absorbance                                                           based on the theoretical                                                      value  (ii)/(iv)!                                                             ______________________________________                                    

It can be seen from these results that, when serum samples are measuredaccording to the present method for the determination of apolipoprotein(a), measured values approximately equal to theoretical ones areobtained.

Thus, it has been confirmed that the method for the determination ofapolipoprotein (a) in accordance with the present invention is a methodcapable of determining apolipoprotein (a) in serum samples accuratelywithout undergoing the influence of nonspecific reactions or the likecaused by the serum samples and, therefore, can be practically used forpurposes of clinical examination.

EXAMPLE 22

Preparation of a mouse monoclonal antibody to lipoprotein (a)specifically recognizing the amino acid sequence represented by SEQ IDNO:2 in the List of Sequences

1! Immunization of an animal

(1) The antibody-producing immunogen (having KLH as the carrier)obtained in Example 7 was dissolved in physiological saline (a 0.9%aqueous solution of sodium chloride) so as to give a concentration of500 μg/ml.

This solution was mixed with an equal amount of Freund's completeadjuvant to form an emulsion, and 0.5 ml of this emulsion wassubcutaneously injected into the abdomen of a female BALB/c mouse(Charles River Japan Inc.) aged 8 weeks for purposes of immunization.

(2) Two weeks after the initial immunization, the above-describedantibody-producing immunogen was dissolved in physiological saline so asto give a concentration of 250 μg/ml, this solution was mixed with anequal amount of Freund's incomplete adjuvant to form an emulsion, and0.5 ml of this emulsion was injected as a booster. This boosterinjection was repeated at intervals of 2 weeks.

(3) The antibody titer in the blood serum of this mouse, which was animmunized animal, was measured by an enzyme immunoassay (ELISA, EIA) atintervals of one week starting from 6 weeks after the initialimmunization. This ELISA was carried out as follows: Theantibody-producing immunogen (having BSA as the carrier) obtained inExample 7 was converted into a solid phase on a microplate, and bloodserum obtained from the immunized animal was added thereto to effectreaction. After washing, peroxidase (POD)-labeled anti-mouse IgGantibody was added to the microplate to effect reaction. After washing,a color-producing solution containing hydrogen peroxide and2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was addedto the microplate to develop color. Then, the antibody titer wasdetermined by measuring the absorbance at 415 nm on an EIA plate reader(manufactured by Bio-Rad).

(4) After the lapse of 16 weeks from the initial immunization, it wasnoted that the antibody titer reached a plateau. Accordingly, theantibody-producing immunogen (having KLH as the carrier) obtained inExample 7 was dissolved in physiological saline so as to give aconcentration of 800 μg/ml, and 0.5 ml of this solution wassubcutaneously injected into the abdomen of this immunized mouse.

After 3 days, the spleen was excised from this immunized mouse.

2! Growth of myeloma cells

The P3-X63-Ag8-U1 strain (Japanese Cancer Research Resources Bank 9085),which is a hypoxanthine-guanine phosphoribosyltransferase-deficientmyeloma cell strain derived from a BALB/c mouse, was grown in RPMI1640tissue culture medium (manufactured by Biocell) containing 10% fetalcalf serum and supplemented with glutamine, penicillin and streptomycin.

More specifically, such myeloma cells were grown in a medium-sizedbottle for cell culture (manufactured by Nunc; 200 ml capacity) untilabout 80% of the bottom surface of the bottle was occupied by the cells.The number of cells was counted with a hemocytometer according to thedye-exclusion technique using trypan blue.

3! Cell fusion

(1) The spleen obtained from the above-described immunized mouse wasfully macerated by use of stainless steel mesh #200 and filtered whilebeing washed with serum-free RPMI1640 liquid medium.

Thereafter, the resulting cell suspension was centrifuged at 200×g toseparate spleen cells.

These spleen cells were further washed three times with serum-freeRPMI1640 liquid medium.

(2) These spleen cells and the above-described grown P3-X63-Ag8-U1strain myeloma cells were mixed in a ratio of 5:1 and centrifuged.

The mixed cells were slowly suspended in RPMI1640 liquid mediumcontaining 50% polyethylene glycol 1500 (PEG 1500; manufactured byBoehringer Mannheim).

Then, this suspension was gradually diluted with RPMI1640 liquid mediumso as to give a final polyethylene glycol concentration of 5%.

(3) The cells were separated therefrom by centrifugation and dispersedslowly in a growth medium comprising S-clone medium (manufactured bySanko Junyaku Co., Ltd.) containing 5% hybridoma cloning factor(manufactured by Origen).

Each well of a 96-well flat-bottom micro titer plate (manufactured byNunc) was inoculated with 100 μl of the suspension containing 10⁶ cells,and incubated at 37° C. in an atmosphere containing 5% carbon dioxide.

(4) One day after the cell fusion, 100 μl of HAT medium theabove-described growth medium supplemented with 0.01 mM hypoxanthine,1.6 μM thymidine and 0.04 μM aminopterin (all manufactured by TokyoKasei Kogyo Co., Ltd.)! was added to each well.

For the succeeding 3 days, about a half of the HAT medium was replacedevery day by fresh HAT medium. Thereafter, the same replacement wasperformed at intervals of 2 or 3 days.

(5) Cells were observed under the microscope.

Clones of hybridomas (fused cells) began to appear after 10 days ormore. After 14 days or more, the supernatants obtained from the wellswere screened by ELISA in order to test them for the production of anantibody recognizing the amino acid sequence represented by SEQ ID NO:2in the List of Sequences. This ELISA was carried out in the same manneras in Reference Example 2.

(6) With respect to the wells giving a positive test for antibodyproduction, the cells were spread over a 24-well plate and cultured. Asthe cell density became higher, the cells were cultured on larger scalesusing small-sized and medium-sized bottles.

(7) Hybridomas were cultured and maintained in HT medium (HAT mediumcontaining neither aminopterin nor hybridoma cloning factor).

(8) The production of an antibody recognizing the amino acid sequencerepresented by SEQ ID NO:2 in the List of Sequences was tested by ELISAin the same manner as in Reference Example 2. As a result, there weredetected four hybridomas productive of an antibody combining with theantibody-producing immunogen (having BSA as the carrier) obtained inExample 7, which is a peptide including the amino acid sequencerepresented by SEQ ID NO:2 in the List of Sequence, but not combiningwith BSA.

4! Hybridoma subcloning

(1) The above-described 4 hybridomas were subcloned according to thelimiting dilution technique.

The number of cells of each hybridoma was counted with a hemocytometeraccording to the dye-exclusion technique using trypan blue.

Then, the cells of each hybridoma were suspended in HT medium at twocell densities of 0.5 and 1 viable cell per 100 μl of HT medium. Each ofthese suspensions was pipetted into the wells of a 96-well flat-bottommicroplate in an amount of 100 μl per well.

These hybridomas were grown with the medium being replaced at intervalsof 2 or 3 days.

(2) After 2 weeks, the number of colonies present in each well wascounted under the microscope, and the wells productive of an antibodycombining with the antibody-producing immunogen (having BSA as thecarrier) obtained in Example 7, which is a peptide including the aminoacid sequence represented by SEQ ID NO:2 in the List of Sequence, butnot combining with BSA were detected by ELISA in the same manner asdescribed above.

Thus, there were obtained four wells which each contained only onecolony and were productive of an antibody as described above.

(3) These hybridomas were transferred to a 24-well plate and culturedfor 2 weeks until a good growth of cells was achieved.

(4) Then, the reactivity with lipoprotein (a) of the antibodies producedby these hybridomas was tested by the western blot technique.

This western blot technique was carried out in the same manner as inReference Example S, except that their reactivity with LDL was nottested and the supernatants of the hybridoma cultures were used in placeof the polyclonal antibodies to lipoprotein (a) obtained in Examples 9and 10.

As a result, one hybridoma was found to be a cell strain productive ofan antibody to lipoprotein (a) specifically recognizing the amino acidsequence represented by SEQ ID NO:2 in the List of Sequences.

(5) This hybridoma was cloned again in the same manner as describedabove in steps (1) and (2). By examining each well for antibodyproduction, there were obtained a total of 40 hybridoma clonescharacterized in that only one hybridoma colony was present in a welland the hybridoma colony which produced an antibody combining with theantibody-producing immunogen (having BSA as the carrier) obtained inExample 7, which is a peptide including the amino acid sequencerepresented by SEQ ID NO:2 in the List of Sequences, but not combiningwith BSA.

(6) The reactivity with lipoprotein (a) of the antibodies produced bythese hybridoma clones was tested again by the western blot technique inthe same manner as described above in step (4).

As a result, it has been confirmed that all of these hybridoma clonesare cells productive of an antibody to lipoprotein (a) whichspecifically recognizes the amino acid sequence represented by SEQ IDNO:2 in the List of Sequences.

(7) This cell strain was employed as a hybridoma cell strain (243G7E7F10strain) for producing a monoclonal antibody to lipoprotein (a) whichspecifically recognizes a part or the whole of the amino acid sequencerepresented by SEQ ID NO:2 in the List of Sequences.

This hybridoma cell strain (243G7E7F10 strain) was deposited on Aug. 4,1993 with the National Institute of Bioscience and Human-Technology, theAgency of Industrial Science and Technology, the Ministry ofInternational Trade and Industry, Japan, and assigned FERM BP-4379.

5! Production of a monoclonal antibody

(1) Using a medium-sized bottle (manufactured by Nunc), the resultingcell strain for producing a monoclonal antibody to lipoprotein (a) wasgrown in HT medium until about 80% of its bottom surface was occupied bycells.

(2) Thereafter, these hybridoma cells were scraped out and collected bycentrifugation at 200×g for 5 minutes.

Subsequently, they were washed three times with serum-free RPMI1640liquid medium and then suspended in 2 ml of RPMI1640 liquid medium.

(3) 1 ml of the hybridoma cell suspension obtained in theabove-described step (2) was injected into the abdominal cavity of amale BALB/c mouse (Charles River Japan Inc.) which had previously beentreated with 2,6,10,14-tetramethylpentadecane.

If no expansion of the abdomen of this mouse was observed within 2 weeksafter the injection, the same procedure was repeated.

(4) As soon as an expansion of the abdomen of this mouse was observed,ascites was collected therefrom.

This ascites was centrifuged at 200×g for 5 minutes to separate thesupernatant containing a monoclonal antibody to lipoprotein (a) from thehybridoma cells.

6! Purification of the monoclonal antibody

(1) 1.8 g of sodium sulfate was added, with stirring, at 22° C. to 10 mlof the supernatant containing a monoclonal antibody to lipoprotein (a).After sodium sulfate was completely dissolved, the stirring wascontinued for an additional hour to effect salting-out.

(2) This mixture was centrifuged at 22° C. (7000×g, 15 minutes), and theprecipitate separated from the supernatant was dissolved in 2 ml of 40mM sodium phosphate buffer (pH 8.0) containing 30 mM sodium chloride.

(3) Next, this solution was thoroughly dialyzed against 40 mM sodiumphosphate buffer (pH 8.0) containing 30 mM sodium chloride, and thencentrifuged at 1000×g for 20 minutes to remove any insoluble matter.

(4) This solution was passed at a flow rate of 0.4 ml/min. through aDEAE-cellulose ion exchange column (1×10 cm; manufactured by Serva)which had been equilibrated with 40 mM sodium phosphate buffer (pH 8.0)containing 30 mM sodium chloride, and the eluate was collected in 2 mlfractions.

(5) Absorbance measurement at 280 nm revealed that immunoglobulin G(IgG) was contained in the flow-through fractions of the eluate.Accordingly, the flow-through fractions were collected and concentratedto 2 ml.

(6) The resulting concentrate was further purified by proteinA-Sepharose CL-4B affinity chromatography (manufactured byPharmacia-LKB) to obtain a mouse monoclonal antibody to lipoprotein (a)specifically recognizing the amino acid sequence represented by SEQ IDNO:2 in the List of Sequences.

The amount of the monoclonal antibody thus obtained was 15 mg asexpressed in terms of protein.

According to the Ouchterlony immunodiffusion method using a commerciallyavailable specific anti-mouse immunoglobulin antiserum (manufactured byDako), the class and subtype of the resulting monoclonal antibody tolipoprotein (a) was determined to be IgG₁ and λ chain respectively.

Reference Example 10

Reactivity of the monoclonal antibody to lipoprotein (a) obtained inExample 22 with the peptides represented by SEQ ID NO:8, SEQ ID NO:9 andSEQ ID NO:10 in the List of Sequences

The reactivity of the monoclonal antibody to lipoprotein (a) obtained inExample 22 with the peptides represented by SEQ ID NO:8, SEQ ID NO:9 andSEQ ID NO:10 in the List of Sequences, which are peptides including theamino acid sequences represented by SEQ ID NO:1, SEQ ID NO:2 and SEQ IDNO:3 in the List of Sequences, respectively, was confirmed by ELISA.

(1) The antibody-producing immunogen (having BSA as the carrier)obtained in Example 6, the antibody-producing immunogen (having BSA asthe carrier) obtained in Example 7, and the antibody-producing immunogen(having BSA as the carrier) obtained in Example 8 were each dissolved inphysiological saline (a 0.9% aqueous solution of sodium chloride) so asto give a concentration of 5 μg/ml. These solutions were added to wellsof a 96-well microplate (manufactured by Nunc) in an amount of 100 μlper well and allowed to stand at 37° C. for 2 hours to convert thepeptides into a solid phase.

(2) After this microplate was washed with a washing solutionphosphate-buffered physiological saline (5.59 mM disodium hydrogenphosphate, 1.47 mM potassium dihydrogen phosphate, 137 mM sodiumchloride, 2.68 mM potassium chloride (pH 7.2)) containing 0.05% Tween20!, 10 mM potassium dihydrogen phosphate-dipotassium hydrogen phosphatebuffer (pH 7.2) containing 1% BSA was added to the wells thereof in anamount of 300 μl per well and allowed to stand at 37° C. for 2 hours toeffect blocking. Thereafter, the microplate was washed again with thewashing solution.

(3) The monoclonal antibody to lipoprotein (a) obtained in Example 22was diluted with phosphate-buffered physiological saline containing 3%BSA to prepare four samples having antibody concentrations of 0.1 μg/ml,0.5 μg/ml, 1.0 μg/ml and 5.0 μg/ml, respectively. These dilutions wasadded to the respective wells in an amount of 100 μl per well andallowed to stand at 37° C. for 2 hours to effect reaction. Thereafter,the microplate was washed with the washing solution.

(4) As a control, phosphate-buffered physiological saline containing 3%BSA was added to the wells of another microplate as obtained from step(2) in an amount of 100 μl per well and allowed to stand at 37° C. for 2hours to effect reaction. Thereafter, the microplate was washed with thewashing solution.

(5) Peroxidase (POD)-labeled anti-mouse IgG antibody (manufactured byAmersham) was diluted 2,000-fold with phosphate-buffered physiologicalsaline containing 3% BSA, added to the wells of the microplates obtainedfrom steps (3) and (4) in an amount of 100 μl per well and allowed tostand at 37° C. for 2 hours to effect reaction.

(6) After these microplates were washed with the washing solution, aperoxidase reaction solution (prepared by adding 2 μl of 1.7% hydrogenperoxide to 1 ml of 50 mM disodium hydrogen phosphate-24 mM citric acidbuffer containing 3 mM o-phenylenediamine, immediately before use) wasadded to the wells thereof in an amount of 100 μl per well and they werereacted at room temperature. After 15 minutes, 6N sulfuric acid wasadded to the wells thereof in an amount of 50 μl per well to stop thereaction.

(7) The absorbances at 492 nm of the wells of these microplates weremeasured on an EIA plate reader (manufactured by Bio-Rad).

The results of these measurements are shown in FIG. 26. The data showntherein are those obtained by subtracting the absorbance of the controlfrom the absorbance of each sample.

In FIG. 26, numerals 1, 2 and 3 represent the measured values(absorbances) for the antibody-producing immunogen obtained in Example6, the antibody-producing immunogen obtained in Example 7, and theantibody-producing immunogen obtained in Example 8, respectively.

From these results, it has been confirmed that the monoclonal antibodyto lipoprotein (a) obtained in Example 22 does not combine with thepeptides represented by SEQ ID NO:8 and SEQ ID NO:10 in the List ofSequences which are peptides including the amino acid sequencesrepresented by SEQ ID NO:1 and SEQ ID NO:3 in the List of Sequences,respectively, but specifically recognizes and combines with the peptiderepresented by SEQ ID NO:9 in the List of Sequences which is a peptideincluding the amino acid sequence represented by SEQ ID NO:2 in the Listof Sequences.

EXAMPLE 23

Preparation of a mouse monoclonal antibody to apolipoprotein (a)specifically recognizing the amino acid sequence represented by SEQ IDNO:3 in the List of Sequences

1! Immunization of an animal

(1) The antibody-producing immunogen (having KLH as the carrier)obtained in Example 8 was dissolved in physiological saline (a 0.9%aqueous solution of sodium chloride) so as to give a concentration of400 μg/ml. This solution was mixed with an equal amount of Freund'scomplete adjuvant to form an emulsion, and 0.5 ml of this emulsion wassubcutaneously injected into the abdomen of a female BALB/c mouse(Charles River Japan Inc.) aged 8 weeks for purposes of immunization.

(2) Two weeks after the initial immunization, the above-describedantibody-producing immunogen was dissolved in physiological saline so asto give a concentration of 200 μg/ml, this solution was mixed with anequal amount of Freund's incomplete adjuvant to form an emulsion, and0.5 ml of this emulsion was injected as a booster.

This booster injection was repeated at intervals of 2 weeks.

(3) The antibody titer in the blood serum of this mouse, which was animmunized animal, was measured by an enzyme immunoassay (ELISA, EIA) atintervals of one week starting from 6 weeks after the initialimmunization. This ELISA was carried out as follows: Theantibody-producing immunogen (having BSA as the carrier) obtained inExample 8 was converted into a solid phase on a microplate, and bloodserum obtained from the immunized animal was added thereto to effectreaction. After washing, peroxidase (POD)-labeled anti-mouse IgGantibody was added to the microplate to effect reaction. After washing,a color-producing solution containing hydrogen peroxide and2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) was addedto the microplate to develop color. Then, the antibody titer wasdetermined by measuring the absorbance at 415 nm on an EIA plate reader(manufactured by Bio-Rad).

(4) After the lapse of 18 weeks from the initial immunization, it wasnoted that the antibody titer reached a plateau. Accordingly, theantibody-producing immunogen (having KLH as the carrier) obtained inExample 8 was dissolved in physiological saline so as to give aconcentration of 800 μg/ml, and 0.5 ml of this solution wassubcutaneously injected into the abdomen of this immunized mouse.

After 3 days, the spleen was excised from this immunized mouse.

2! Growth of myeloma cells

The P3-X63-Ag8-U1 strain (Japanese Cancer Research Resources Bank 9085),which is a hypoxanthine-guanine phosphoribosyltransferase-deficientmyeloma cell strain derived from a BALB/c mouse, was grown in RPMI1640tissue culture medium (manufactured by Biocell) containing 10% fetalcalf serum and supplemented with glutamine, penicillin and streptomycin.

More specifically, such myeloma cells were grown in a medium-sizedbottle for cell culture (manufactured by Nunc; 200 ml capacity) untilabout 80% of the bottom surface of the bottle was occupied by the cells.The number of cells was counted with a hemocytometer according to thedye-exclusion technique using trypan blue.

3! Cell fusion

(1) The spleen obtained from the above-described immunized mouse wasfully macerated by use of stainless steel mesh #200 and filtered whilebeing washed with serum-free RPMI1640 liquid medium.

Thereafter, the resulting cell suspension was centrifuged at 200×g toseparate spleen cells.

These spleen cells were further washed three times with serum-freeRPMI1640 liquid medium.

(2) These spleen cells and the above-described grown P3-X63-Ag8-U1strain myeloma cells were mixed in a ratio of 5:1 and centrifuged.

The mixed cells were slowly suspended in RPMI1640 liquid mediumcontaining 50% polyethylene glycol 1500 (PEG 1500; manufactured byBoehringer Mannheim).

Then, this suspension was gradually diluted with RPMI1640 liquid mediumso as to give a final polyethylene glycol concentration of 5%.

(3) The cells were separated therefrom by centrifugation and dispersedslowly in a growth medium comprising S-clone medium (manufactured bySanko Junyaku Co., Ltd.) containing 5% hybridoma cloning factor(manufactured by Origen).

Each well of a 96-well flat-bottom micro titer plate (manufactured byNunc) was inoculated with 100 11 of the suspension containing 10⁶ cells,and incubated at 37° C. in an atmosphere containing 5% carbon dioxide.

(4) One day after the cell fusion, 100 μl of HAT medium theabove-described growth medium supplemented with 0.01 mM hypoxanthine,1.6 μM thymidine and 0.04 μM aminopterin (all manufactured by TokyoKasei Kogyo Co., Ltd.)! was added to each well.

For the succeeding 3 days, about a half of the HAT medium was replacedevery day by fresh HAT medium. Thereafter, the same replacement wasperformed at intervals of 2 or 3 days.

(5) Cells were observed under the microscope.

Clones of hybridomas (fused cells) began to appear after 10 days ormore. After 14 days or more, the supernatants obtained from the wellswere screened by ELISA in order to test them for the production of anantibody recognizing the amino acid sequence represented by SEQ ID NO:3in the List of Sequences. This ELISA was carried out in the same manneras in Reference Example 5.

(6) With respect to the wells giving a positive test for antibodyproduction, the cells were spread over a 24-well plate and cultured. Asthe cell density became higher, the cells were cultured on larger scalesusing small-sized and medium-sized bottles.

(7) Hybridomas were cultured and maintained in HT medium (HAT mediumcontaining neither aminopterin nor hybridoma cloning factor).

(8) The production of an antibody recognizing the amino acid sequencerepresented by SEQ ID NO:3 in the List of Sequences was tested by ELISAin the same manner as in Reference Example 5. As a result, there weredetected four hybridomas productive of an antibody combining with theantibody-producing immunogen (having BSA as the carrier) obtained inExample 8, which is a peptide including the amino acid sequencerepresented by SEQ ID NO:3 in the List of Sequence, but not combiningwith BSA.

4! Hybridoma subcloning

(1) The above-described 4 hybridomas were subcloned according to thelimiting dilution technique.

The number of cells of each hybridoma was counted with a hemocytometeraccording to the dye-exclusion technique using trypan blue.

Then, the cells of each hybridoma were suspended in HT medium at twocell densities of 0.5 and 1 viable cell per 100 μl of HT medium. Each ofthese suspensions was pipetted into the wells of a 96-well flat-bottommicroplate in an amount of 100 μl per well.

These hybridomas were grown with the medium being replaced at intervalsof 2 or 3 days.

(2) After 2 weeks, the number of colonies present in each well wascounted under the microscope, and the wells productive of an antibodycombining with the antibody-producing immunogen (having BSA as thecarrier) obtained in Example 8, which is a peptide including the aminoacid sequence represented by SEQ ID NO:3 in the List of Sequence, butnot combining with BSA were detected by ELISA in the same manner asdescribed above.

Thus, there were obtained four wells which each contained only onecolony and were productive of an antibody as described above.

(3) These hybridomas were transferred to a 24-well plate and culturedfor 2 weeks until a good growth of cells was achieved.

(4) Then, the reactivity with apolipoprotein (a) of the antibodiesproduced by these hybridomas was tested by the western blot technique.

This western blot technique was carried out in the same manner as inReference Example 6, except that only one type of purifiedapolipoprotein (a) was subjected to electrophoresis in step (2) ofReference Example 6, and the supernatants of the hybridoma cultures wereused in place of the polyclonal antibody to apolipoprotein (a) obtainedin Examples 11.

As a result, one hybridoma was found to be a cell strain productive ofan antibody to apolipoprotein (a) specifically recognizing the aminoacid sequence represented by SEQ ID NO:3 in the List of Sequences.

(5) This hybridoma was cloned again in the same manner as describedabove in steps (1) and (2). By examining each well for antibodyproduction, there were obtained a total of 15 hybridoma clonescharacterized in that only one hybridoma colony was present in a welland the hybridoma colony which produced an antibody combining with theantibody-producing immunogen (having BSA as the carrier) obtained inExample 8, which is a peptide including the amino acid sequencerepresented by SEQ ID NO:3 in the List of Sequences, but not combiningwith BSA.

(6) The reactivity with apolipoprotein (a) of the antibodies produced bythese hybridoma clones was tested again by the western blot technique inthe same manner as described above in step (4).

As a result, it has been confirmed that all of these hybridoma clonesare cells productive of an antibody to apolipoprotein (a) whichspecifically recognizes the amino acid sequence represented by SEQ IDNO:3 in the List of Sequences.

(7) This cell strain was employed as a hydrodoma cell strain (161E2H6strain) for producing a monoclonal antibody to apolipoprotein (a) whichspecifically recognizes a part or the whole of the amino acid sequencerepresented by SEQ ID NO:3 in the List of Sequences.

This hydrodoma cell strain (161E2H6 strain) was deposited on Aug. 4,1993 with the National Institute of Bioscience and Human-Technology, theAgency of Industrial Science and Technology, the Ministry ofInternational Trade and Industry, Japan, and assigned FERM BP-4378.

5! Production of a monoclonal antibody

(1) Using a medium-sized bottle (manufactured by Nunc), the resultingcell strain for producing a monoclonal antibody to apolipoprotein (a)was grown in HT medium until about 80% of its bottom surface wasoccupied by cells.

(2) Thereafter, these hybridoma cells were scraped out and collected bycentrifugation at 200×g for 5 minutes.

Subsequently, they were washed three times with serum-free RPMI1640liquid medium and then suspended in 2 ml of RPMI1640 liquid medium.

(3) 1 ml of the hybridoma cell suspension obtained in theabove-described step (2) was injected into the abdominal cavity of amale BALB/c mouse (Charles River Japan Inc.) which had previously beentreated with 2, 6, 10, 14-tetramethyl-pentadecane.

If no expansion of the abdomen of this mouse was observed within 2 weeksafter the injection, the same procedure was repeated.

(4) As soon as an expansion of the abdomen of this mouse was observed,ascites was collected therefrom.

This ascites was centrifuged at 200×g for 5 minutes to separate thesupernatant containing a monoclonal antibody to apolipoprotein (a) fromthe hybridoma cells.

6! Purification of the monoclonal antibody

(1) 1.8 g of sodium sulfate was added, with stirring, at 22° C. to 10 mlof the supernatant containing a monoclonal antibody to apolipoprotein(a). After sodium sulfate was completely dissolved, the stirring wascontinued for an additional hour to effect salting-out.

(2) This mixture was centrifuged at 22° C. (7000×g, 15 minutes), and theprecipitate separated from the supernatant was dissolved in 2 ml of 40mM sodium phosphate buffer (pH 8.0) containing 30 mM sodium chloride.

(3) Next, this solution was thoroughly dialyzed against 40 mM sodiumphosphate buffer (pH 8.0) containing 30 mM sodium chloride, and thencentrifuged at 1000×g for 20 minutes to remove any insoluble matter.

(4) This solution was passed at a flow rate of 0.4 ml/min. through aDEAE-cellulose ion exchange column (1×10 cm; manufactured by Serva)which had been equilibrated with 40 mM sodium phosphate buffer (pH 8.0)containing 30 mM sodium chloride, and the eluate was collected in 2 mlfractions.

(5) Absorbance measurement at 280 nm revealed that immunoglobulin G(IgG) was contained in the flow-through fractions of the eluate.Accordingly, the flow-through fractions were collected and concentratedto 2 ml.

(6) The resulting concentrate was further purified by proteinA-Sepharose CL-4B affinity chromatography (manufactured byPharmacia-LKB) to obtain a mouse monoclonal antibody to apolipoprotein(a) specifically recognizing the amino acid sequence represented by SEQID NO:3 in the List of Sequences.

The amount of the monoclonal antibody thus obtained was 10 mg asexpressed in terms of protein.

According to the Ouchterlony immunodiffusion method using a commerciallyavailable specific anti-mouse immunoglobulin antiserum (manufactured byDako), the class and subtype of the resulting monoclonal antibody toapolipoprotein (a) was determined to be IgG₁ and λ chain, respectively.

Reference Example 11

Reactivity of the monoclonal antibody to apolipoprotein (a) obtained inExample 23 with the peptides represented by SEQ ID NO:8, SEQ ID NO:9 andSEQ ID NO:10 in the List of Sequences

The reactivity of the monoclonal antibody to apolipoprotein (a) obtainedin Example 23 with the peptides represented by SEQ ID NO:8, SEQ ID NO:9and SEQ ID NO:10 in the List of Sequences, which are peptides includingthe amino acid sequences represented by SEQ ID NO:1, SEQ ID NO:2 and SEQID NO:3 in the List of Sequences, respectively, was confirmed by ELISA.

Measurements were made in the same manner as in Reference Example 10,except that the monoclonal antibody to apolipoprotein (a) obtained inExample 23 was used in place of the monoclonal antibody to lipoprotein(a) obtained in Example 22, and four samples of this antibody hadconcentrations of 5.0 μg/ml, 10 μg/ml, 50 μg/ml and 100 μ/ml,respectively.

The results of these measurements are shown in FIG. 27. The data showntherein are those obtained by subtracting the absorbance of the controlfrom the absorbance of each sample.

In FIG. 27, numerals 1, 2 and 3 represent the measured values(absorbances) for the antibody-producing immunogen obtained in Example6, the antibody-producing immunogen obtained in Example 7, and theantibody-producing immunogen obtained in Example 8, respectively.

From these results, it has been confirmed that the monoclonal antibodyto lipoprotein (a) obtained in Example 23 does not combine with thepeptides represented by SEQ ID NO:8 and SEQ ID NO:9 in the List ofSequences which are peptides including the amino acid sequencesrepresented by SEQ ID NO:1 and SEQ ID NO:2 in the List of Sequences,respectively, but specifically recognizes and combines with the peptiderepresented by SEQ ID NO:10 in the List of Sequences which is a peptideincluding the amino acid sequence represented by SEQ ID NO:3 in the Listof Sequences.

Reference Example 12

Reactivity with apolipoprotein (a) of the monoclonal antibody toapolipoprotein (a) obtained in Example 23

The reactivity with apolipoprotein (a) of the monoclonal antibody toapolipoprotein (a) obtained in Example 23 was confirmed by the westernblot technique.

The western blot technique was carried out in the same manner as inReference Example 6, except that five types of purified apolipoprotein(a) derived from human blood serum were subjected to electrophoresis,and the monoclonal antibody to apolipoprotein (a) obtained in Example 23was used in place of the polyclonal antibody to apolipoprotein (a)obtained in Example 11.

The results of this western blot technique are shown in FIGS. 28, 29 and30.

FIG. 28 shows the nitrocellulose membrane acted on by the monoclonalantibody to apolipoprotein (a) obtained in Example 23.

In FIG. 28, numerals 1 to 5 represent the respective electrophoreticpatterns of the five types of purified apolipoprotein (a) derived fromhuman blood serum, and B shows the position of the band ofapolipoprotein B-100 useful as a reference standard for identifying theresulting electrophoretic bands.

On the basis of positional relationship with this band of apolipoproteinB-100 G. Utermann et al., J. Clin. Invest., 80, 458-465(1987)!, it canbe seen that the bands observed in patterns 1 to 5 represent F, B, S1,S2, S3 and S4 isotypes of apolipoprotein (a).

FIG. 29 shows the negative control in which numerals 1 to 5 representthe respective electrophoretic patterns of the five types of purifiedapolipoprotein (a) derived from human blood serum. R represents, as areference standard, the electrophoretic pattern of the purifiedapolipoprotein (a) derived blood serum as represented above by numeral1.

FIG. 30 shows the control in which the nitrocellulose membrane was actedon by anti-lipoprotein (a) antibody reacting with apolipoprotein (a).The sample used was the purified apolipoprotein (a) derived from bloodserum which corresponds to the above-described pattern 1. B shows theposition of the band of apolipoprotein B-100 as a reference standard.

As can be seen from FIGS. 28 and 30, the monoclonal antibody toapolipoprotein (a) obtained in Example 23 exhibits color development atthe same position as anti-lipoprotein (a) antibody reacting withapolipoprotein (a) does. Thus, it has been confirmed that thismonoclonal antibody combines specifically with apolipoprotein (a).

Moreover, in FIG. 28, it has been confirmed that the monoclonal antibodyto apolipoprotein (a) obtained in Example 23 react with various isotypesof apolipoprotein (a).

Furthermore, in FIG. 29, no color development is observed in thenegative control which was not acted on by the monoclonal antibody toapolipoprotein (a) obtained in Example 23 or anti-lipoprotein (a)antibody reacting with apolipoprotein (a), indicating that nonspecificcolor development did not take place.

Reference Example 13

Reactivity with lipoprotein (a) of the monoclonal antibody tolipoprotein (a) obtained in Example 22 and the monoclonal antibody toapolipoprotein (a) obtained in

EXAMPLE 23

The reactivity with lipoprotein (a) of each of the monoclonal antibodyto lipoprotein (a) obtained in Example 22 and the monoclonal antibody toapolipoprotein (a) obtained in Example 23 was confirmed by the westernblot technique.

The western blot technique was carried out in the same manner as inReference Example 7, except that each of the monoclonal antibody tolipoprotein (a) obtained in Example 22 and the monoclonal antibody toapolipoprotein (a) obtained in Example 23 was used in place of thepolyclonal antibody to apolipoprotein (a) obtained in Example 11.

The results of this western blot technique are shown in FIG. 31.

In FIG. 31, P represents the control, N represents the negative control,numeral 2 represents the nitrocellulose membrane acted on by themonoclonal antibody to lipoprotein (a) obtained in Example 22, andnumeral 3 represents the nitrocellulose membrane acted on by themonoclonal antibody to apolipoprotein (a) obtained in Example 23.

As can seen by comparison with the control in FIG. 31, the monoclonalantibody to lipoprotein (a) obtained in Example 22 exhibits colordevelopment at the same position as the commercially availableanti-lipoprotein (a) antibody does. Thus, it has been confirmed thatthis monoclonal antibody combines specifically with lipoprotein (a).

Moreover, the monoclonal antibody to apolipoprotein (a) obtained inExample 23 exhibits no color development at the position where thecommercially available anti-lipoprotein (a) antibody exhibits colordevelopment. Thus, it has been confirmed that this monoclonal antibodydoes not combine with lipoprotein (a).

Furthermore, no color development is observed in the negative controlwhich was not acted on by any of the antibody obtained in Example 22,the antibody obtained in Example 23, and the commercially availableanti-lipoprotein (a) antibody, indicating that nonspecific colordevelopment did not take place.

Reference Example 14

Reactivity with LDL of the monoclonal antibody to lipoprotein (a)obtained in Example 22 and the monoclonal antibody to apolipoprotein (a)obtained in Example 23

The reactivity with LDL of each of the monoclonal antibody tolipoprotein (a) obtained in Example 22 and the monoclonal antibody toapolipoprotein (a) obtained in Example 23 was confirmed by the westernblot technique.

(1) Human blood serum having a high LDL concentration wasultracentrifuged to separate a fraction having a specific gravity rangeof 1.006 to 1.063. Then, this serum fraction was subjected to affinitychromatography using anti-lipoprotein (a) antibody (manufactured byImmuno) as the ligand. The flow-through fractions were collected toobtain purified LDL.

(2) This LDL was dissolved in physiological saline (a 0.9% aqueoussolution of sodium chloride) so as to give a concentration of 0.5 mg/ml.A 2 μl sample of this solution was subjected to electrophoresis using aTitan Gel Lipoprotein Electrophoresis Kit (manufactured by HelenaLaboratory). The supporting medium was agarose gel. Using a barbitalbuffer (pH 8.8) as the electrophoresis buffer, a voltage of 90 V wasapplied for 75 minutes.

(3) Using a Nova Blot Electrophoretic Transfer Kit (manufactured byPharmacia-LKB), transfer was carried out on a dry basis according to itsinstruction manual.

(4) The agarose gel obtained from step (2) was placed on a transferapparatus and a 9 cm×9 cm nitrocellulose membrane (manufactured byBio-Rad) was laid thereon. Using a transfer buffer comprising 48 mMTris, 39 mM glycine, 0.0375% (W/V) sodium dodecyl sulfate (SDS) and 20%(V/V) methanol, transfer was carried out by passing an electric currentof 65 mA for 2 hours.

(5) The nitrocellulose membrane having undergone the transfer was soakedovernight in 20 ml of phosphate-buffered physiological saline 5.59 mMdisodium hydrogen phosphate, 1.47 mM potassium dihydrogen phosphate, 137mM sodium chloride, 2.68 mM potassium chloride (pH 7.2)! containing 1%BSA at 4° C. to effect blocking.

(6) Then, this nitrocellulose membrane was washed by shaking it in 20 mlof a washing solution (phosphate-buffered physiological salinecontaining 0.05% Tween 20) for 10 minutes. This procedure was repeatedthree times.

(7) 80 μg each of the monoclonal antibody to lipoprotein (a) obtained inExample 22 and the monoclonal antibody to apolipoprotein (a) obtained inExample 23 was dissolved in 20 ml of phosphate-buffered physiologicalsaline. Two nitro-cellulose membranes as obtained from step (6) wereseparately soaked in these two solutions at room temperature for 2 hoursto effect reaction.

(8) As a control, the procedure described above in step (7) was repeatedby using goat anti-apolipoprotein B antibody to apolipoprotein B-100being a component of LDL (manufactured by international Enzyme),dissolved at the same concentration, in place of the monoclonal antibodyto lipoprotein (a) obtained in Example 22 and the monoclonal antibody toapolipoprotein (a) obtained in Example 23.

In addition, another nitrocellulose membrane as obtained from step (6),which was not acted on by any of the monoclonal antibody to lipoprotein(a) obtained in Example 22, the monoclonalantibody to apolipoprotein (a)obtained in Example 23, and goatantiapolipoprotein B antibody, wasprovided as a negative control.

(9) The nitrocellulose membranes subjected to the procedure describedabove in step (7) or (8) were washed by shaking them in 20 ml of thewashing solution for 10 minutes. This procedure was repeated threetimes.

(10) Next, peroxidase-labeled anti-mouse IgG antibody (manufacture byDako) and peroxidase-labeled anti-goat IgG antibody (manufactured byDako) were diluted 500-fold with phosphate-buffered physiological salinecontaining 3% BSA to prepare 20 ml of a solution, and the nitrocellulosemembranes were soaked therein at room temperature for 2 hours to effectreaction.

(11) These nitrocellulose membranes were washed by shaking them in 20 mlof the washing solution for 10 minutes. This procedure was repeatedthree times.

(12) The nitrocellulose membranes obtained from step (11) were soaked in20 ml of phosphate-buffered physiological saline containing 0.025%3,3'-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide atroom temperature for 15 minutes to develop color.

The results of this western blot technique are shown in FIG. 32.

In FIG. 32, P represents the control, N represents the negative control,numeral 2 represents the nitrocellulose membrane acted on by themonoclonal antibody to lipoprotein (a) obtained in Example 22, andnumeral 3 represents the nitrocellulose membrane acted on by themonoclonal antibody to apolipoprotein (a) obtained in Example 23.

As can seen by comparison with the control in FIG. 32, the monoclonalantibody to lipoprotein (a) obtained in Example 22 and the monoclonalantibody to apolipoprotein (a) obtained in Example 23 exhibit no colordevelopment at the position where the commercially availableanti-apolipoprotein B antibody exhibits color development. Thus, it hasbeen confirmed that these monoclonal antibodies do not combine with LDL.

Furthermore, no color development is observed in the negative controlwhich was not acted on by any of the monoclonal antibody obtained inExample 22, the monoclonal antibody obtained in Example 23, and thecommercially available anti-apolipoprotein B antibody, indicating thatnonspecific color development did not take place.

Reference Example 15

Reactivity with plasminogen of the monoclonal antibody to lipoprotein(a) obtained in Example 22 and the monoclonal antibody to apolipoprotein(a) obtained in Example 23

The reactivity with plasminogen of each of the monoclonal antibody tolipoprotein (a) obtained in Example 22 and the monoclonal antibody toapolipoprotein (a) obtained in Example 23 was confirmed by the westernblot technique.

The western blot technique was carried out in the same manner as inReference Example 4, except that the monoclonal antibody to lipoprotein(a) obtained in Example 22 and the monoclonal antibody to apolipoprotein(a) obtained in Example 23 were used in place of the polyclonalantibodies to lipoprotein (a) obtained in Examples 9 and 10,respectively.

The results of this western blot technique are shown in FIG. 33.

In FIG. 33, P represents the control, N represents the negative control,numeral 2 represents the nitrocellulose membrane acted on by themonoclonal antibody to lipoprotein (a) obtained in Example 22, andnumeral 3 represents the nitrocellulose membrane acted on by themonoclonal antibody to apolipoprotein (a) obtained in Example 23.

As can seen from these results, the monoclonal antibody to lipoprotein(a) obtained in Example 22 and the monoclonal antibody to apolipoprotein(a) obtained in Example 23 exhibit no color development at the positionwhere the commercially available anti-plasminogen antibody exhibitscolor development. Thus, it has been confirmed that these monoclonalantibodies do not combine with plasminogen.

Furthermore, no color development is observed in the negative controlwhich was not acted on by the monoclonal antibody obtained in Example22, the monoclonal antibody obtained in Example 23, or the like,indicating that nonspecific color development did not take place.

EXAMPLE 24

Method for the determination of lipoprotein (a) by ELISA

A system for the determination of lipoprotein (a) by ELISA wasestablished using the monoclonal antibody to lipoprotein (a) obtained inExample 22.

Measurements were made in the same manner as in Example 17, except thatthe monoclonal antibody to lipoprotein (a) obtained in Example 22 wasused at the same concentration in step (5) of Example 17, in place ofthe polyclonal antibody to lipoprotein (a) obtained in Example 9.

The calibration curve obtained by measuring five samples is shown inFIG. 34.

From these results, it has been confirmed that the method for thedetermination of lipoprotein (a) in accordance with the presentinvention enables quantitative determination of lipoprotein (a).

EXAMPLE 25

Comparison of the measured values for lipoprotein (a)

The measured values obtained by the present method for the determinationof lipoprotein (a) by ELISA using the monoclonal antibody to lipoprotein(a) obtained in Example 22 were compared with those obtained by ELISAusing the lipoprotein (a)-determining reagent of company A.

Samples 1 to 10 comprising ten types of blood sere were measuredaccording to the present method for the determination of lipoprotein (a)by ELISA using the monoclonal antibody to lipoprotein (a) obtained inExample 22. The ELISA was carried out in the same manner as in Example17.

The measurement of ten samples by using the lipoprotein (a)-determiningreagent of company A was made according to its instruction manual.

The results of these measurements are summarized in Table 6.

                  TABLE 6                                                         ______________________________________                                                   Method of the                                                                          Reagent of                                                           invention                                                                              company A                                                 ______________________________________                                        Sample 1     6.5        8.0                                                   Sample 2     2.2        3.7                                                   Sample 3     33.5       31.0                                                  Sample 4     12.5       10.9                                                  Sample 5     14.0       12.5                                                  Sample 6     18.0       17.5                                                  Sample 7     6.0        4.5                                                   Sample 8     57.5       54.5                                                  Sample 9     9.3        6.4                                                   Sample 10    75.0       72.9                                                  ______________________________________                                         (The values are expressed in mg/dl.)                                     

From these results, it has been confirmed that the measured values forlipoprotein (a) obtained by the present method for the determination oflipoprotein (a) are substantially the same as those obtained by themethod currently in use and, therefore, the method for the determinationof lipoprotein (a) in accordance with the present invention can bepractically used for purposes of clinical examination.

EXAMPLE 26

Method for the determination of apolipoprotein (a) by ELISA

A system for the determination of apolipoprotein (a) by ELISA wasestablished using the monoclonal antibody to apolipoprotein (a) obtainedin Example 23.

Measurements were made in the same manner as in Example 20, except thatthe monoclonal antibody to apolipoprotein (a) obtained in Example 23 wasused in place of the polyclonal antibody to apolipoprotein (a) obtainedin Example 11, and five samples having apolipoprotein (a) concentrationsof 10 mg/dl, 20 mg/dl, 30 mg/dl, 40 mg/dl and 50 mg/dl was used.

The calibration curve obtained by measuring the five samples is shown inFIG. 35.

From these results, it has been confirmed that the method for thedetermination of apolipoprotein (a) in accordance with the presentinvention enables quantitative determination of apolipoprotein (a).

EXAMPLE 27

Influence of serum samples on the method for the determination ofapolipoprotein (a)

In the method for the determination of apolipoprotein (a) by ELISA usingthe monoclonal antibody to apolipoprotein (a) obtained in Example 23, itwas confirmed by addition and recovery tests that the method was notinfluenced by serum samples.

(1) Three types of blood sera (A, B, C) were provided and the followingsamples were prepared using them as base materials.

(i) Three samples prepared by mixing 0.1 ml of physiological saline (a0.9% aqueous solution of sodium chloride) with 0.9 ml of each of thethree blood sera (A, B, C).

(ii) Three samples prepared by diluting the purified apolipoprotein (a)obtained in Example 20 with physiological saline so as to give aconcentration of 100 mg/dl and mixing 0.1 ml of this solution with 0.9ml of each of the three blood sera (A, B, C) to increase theapolipoprotein (a) concentrations of the three serum samples prepared in(i) by 10 mg/ml.

(iii) A sample prepared by diluting the purified apolipoprotein (a)obtained in Example 20 with physiological saline so as to give aconcentration of 10 mg/dl.

(2) The absorbances of the above-described seven samples were measuredaccording to the method for the determination of apolipoprotein (a) byELISA as described in Example 26. The results thus obtained are shown inTable 7.

                  TABLE 7                                                         ______________________________________                                                        Serum A                                                                              Serum B  Serum C                                       ______________________________________                                        (i) Absorbance of each                                                                          0.087    0.109    0.065                                     sample comprising a                                                           blood serum mixed with                                                        physiological saline                                                          (iii) Absorbance of                                                                             0.168    0.168    0.168                                     10 mg/dl of purified                                                          apolipoprotein (a)                                                            (iv) Theoretical absorb-                                                                        0.255    0.277    0.233                                     ance of each serum                                                            sample of (i) having                                                          an apolipoprotein (a)                                                         concentration                                                                 increased by 10 mg/dl                                                          (i) + (iii)!                                                                 (ii) Measured absorbance                                                                        0.244    0.275    0.242                                     of each serum sample                                                          of (i) having an apo-                                                         lipoprotein (a) concen-                                                       tration increased by                                                          10 mg/dl                                                                      (v) Percentage of the                                                                           95.7%    99.3%    104%                                      measured absorbance                                                           based on the theoretical                                                      value  (ii)/(iv)!                                                             ______________________________________                                    

It can be seen from these results that, when serum samples are measuredaccording to the present method for the determination of apolipoprotein(a), measured values approximately equal to theoretical ones areobtained.

Thus, it has been confirmed that the method for the determination ofapolipoprotein (a) in accordance with the present invention is a methodcapable of determining apolipoprotein (a) in serum samples accuratelywithout undergoing the influence of nonspecific reactions or the likecaused by the serum samples and, therefore, can be practically used forpurposes of clinical examination.

Exploitability in Industry

The antibodies to lipoprotein (a) in accordance with the presentinvention are antibodies specifically recognizing lipoprotein (a)without showing a cross reaction with LDL or plasminogen. Accordingly,they do not require troublesome procedures such as ones for absorptiontreatment with LDL or plasminogen and for the selection of a cell strainproductive of an antibody showing no cross reaction with LDL orplasminogen, and hence have the advantage that they can be obtained withless labor, time and cost than prior art antibodies to lipoprotein (a).

Moreover, the peptides selected from the amino acid sequence oflipoprotein (a) in accordance with the present invention and theimmunogens for producing an antibody to lipoprotein (a) in accordancewith the present invention are advantageous in that they do not requirea troublesome and delicate procedure for purification from a biologicalsample and can be stored for a long period of time.

Furthermore, the method for the determination of lipoprotein (a) inaccordance with the present invention is a method capable of determiningthe concentration of lipoprotein (a) accurately without measuringtogether LDL or plasminogen present in the samples.

Similarly, the antibodies to apolipoprotein (a) in accordance with thepresent invention are antibodies specifically recognizing apolipoprotein(a) without showing a cross reaction with lipoprotein (a) orplasminogen. Accordingly, they do not require troublesome proceduressuch as ones for absorption treatment with lipoprotein (a) orplasminogen and for the selection of a cell strain productive of anantibody showing no cross reaction with lipoprotein (a) or plasminogen,and hence have the advantage that they can be obtained with less labor,time and cost than prior art antibodies to apolipoprotein (a).

Moreover, the peptides selected from the amino acid sequence ofapolipoprotein (a) in accordance with the present invention and theimmunogens for producing an antibody to apolipoprotein (a) in accordancewith the present invention are advantageous in that they do not requirea troublesome and delicate procedure for purification from a biologicalsample and can be stored for a long period of time.

Furthermore, the method for the determination of apolipoprotein (a) inaccordance with the present invention is a method capable of determiningthe concentration of apolipoprotein (a) accurately without measuringtogether lipoprotein (a) or plasminogen present in the samples.

    __________________________________________________________________________    SEQUENCE LISTING                                                              (1) GENERAL INFORMATION:                                                      (iii) NUMBER OF SEQUENCES: 10                                                 (2) INFORMATION FOR SEQ ID NO:1:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 8 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                       SerAspAlaGluGlyThrAlaVal                                                      15                                                                            (2) INFORMATION FOR SEQ ID NO:2:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 12 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                       GluAlaProSerGluGlnAlaProThrGluGlnArg                                          1510                                                                          (2) INFORMATION FOR SEQ ID NO:3:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                       ArgAsnProAspAlaValAlaAlaPro                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:4:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 6 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                       AspAlaGluGlyThrAla                                                            15                                                                            (2) INFORMATION FOR SEQ ID NO:5:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 7 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                       GlnAlaProThrGluGlnArg                                                         15                                                                            (2) INFORMATION FOR SEQ ID NO:6:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 5 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                       AlaValAlaAlaPro                                                               15                                                                            (2) INFORMATION FOR SEQ ID NO:7:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 120 amino acids                                                   (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: protein                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                       GluGlnAlaProThrGluGlnArgProGlyValGlnGluCysTyrHis                              151015                                                                        GlyAsnGlyGlnSerTyrArgGlyThrTyrSerThrThrValThrGly                              202530                                                                        ArgThrCysGlnAlaTrpSerSerMetThrProHisSerHisSerArg                              354045                                                                        ThrProGluTyrTyrProAsnAlaGlyLeuIleMetAsnTyrCysArg                              505560                                                                        AsnProAspAlaValAlaAlaProTyrCysTyrThrArgAspProGly                              65707580                                                                      ValArgTrpGluTyrCysAsnLeuThrGlnCysSerAspAlaGluGly                              859095                                                                        ThrAlaValAlaProProThrValThrProValProSerLeuGluAla                              100105110                                                                     ProSerGluGlnAlaProThrGlu                                                      115120                                                                        (2) INFORMATION FOR SEQ ID NO:8:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 9 amino acids                                                     (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                       CysSerAspAlaGluGlyThrAlaVal                                                   15                                                                            (2) INFORMATION FOR SEQ ID NO:9:                                              (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 13 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                       CysGluAlaProSerGluGlnAlaProThrGluGlnArg                                       1510                                                                          (2) INFORMATION FOR SEQ ID NO:10:                                             (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 10 amino acids                                                    (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: unknown                                                         (ii) MOLECULE TYPE: peptide                                                   (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:                                      CysArgAsnProAspAlaValAlaAlaPro                                                1510                                                                          __________________________________________________________________________

We claim:
 1. A peptide composed of 3 to about 50 amino acids andincluding 3 or more consecutive amino acids of an amino acid sequenceselected from SEQ ID NO. 1 and SEQ ID NO. 2, which peptide hasantigenicity as lipoprotein (a) and no antigenicity as low-densitylipoprotein (LDL) or plasminogen.
 2. The peptide according to claim 1,composed of 3 to about 50 amino acids and including 3 or moreconsecutive amino acids of the amino acid sequence represented by SEQ IDNO.
 1. 3. The peptide according to claim 1, composed of 3 to about 50amino acids and including 3 or more consecutive amino acids of the aminoacid sequence represented by SEQ ID NO.
 2. 4. A peptide having an aminoacid sequence selected from the group consisting of SEQ ID NOS. 1, 2, 4,5, 8 and 9, which peptide has antigenicity as lipoprotein (a) and noantigenicity as LDL or plasminogen.
 5. An immunogen for producing anantibody, which comprises a peptide composed of 3 to about 50 aminoacids and including 3 or more consecutive amino acids of an amino acidsequence selected from SEQ ID NO. 1 and SEQ ID NO. 2, which immunogen isuseful to produce an antibody to lipoprotein (a) and has no antigenicityas LDL or plasminogen.
 6. The immunogen for producing an antibody tolipoprotein (a) according to claim 5, which comprises a peptide composedof 3 to about 50 amino acids and including 3 or more consecutive aminoacids of the amino acid sequence represented by SEQ ID NO.
 1. 7. Theimmunogen according to claim 6, wherein the peptide is combined with acarrier.
 8. The immunogen for producing an antibody to lipoprotein (a)according to claim 5, which comprises a peptide composed of 3 to about50 amino acids and including 3 or more consecutive amino acids of theamino acid sequence represented by SEQ ID NO.
 2. 9. The immunogenaccording to claim 8, wherein the peptide is combined with a carrier.